Identification and analysis of miRNAs in IR56 rice in response to BPH infestations of different virulence levels.

Rice production and sustainability are challenged by its most dreadful pest, the brown planthopper (Nilaparvata lugens Stål, BPH). Therefore, the studies on rice?BPH interactions and their underlying mechanisms are of high interest. The rice ontogenetic defense, such as the role of microRNAs (miRNAs) has mostly been investigated against the pathogens, with only a few reports existing against the insect infestations. Thus, revealing the involvement of rice miRNAs in response to BPH infestations will be beneficial in understanding these complex interactions. In this study, the small RNA profiling of the IR56 rice in response to separate BPH infestations of varied virulence levels identified the BPH?responsive miRNAs and revealed the differential transcript abundance of several miRNAs during a compatible and incompatible rice?BPH interaction. The miRNA sequence analysis identified 218 known and 28 novel miRNAs distributed in 54 miRNA families. Additionally, 138 and 140 numbers of differentially expressed (DE) miRNAs were identified during the compatible and incompatible interaction, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the target gene candidates of DE miRNAs (including osa?miR2871a?3p, osa?miR172a, osa?miR166a?5p, osa?miR2120, and osa?miR1859) that might be involved in the IR56 rice defense responses against BPH infestation. Conversely, osa?miR530?5p, osa?miR812s, osa?miR2118g, osa?miR156l?5p, osa?miR435 and two of the novel miRNAs, including novel_16 and novel_52 might negatively modulate the IR56 rice defense. The expressional validation of the selected miRNAs and their targets further supported the IR56 rice defense regulatory network. Based on our results, we have proposed a conceptual model depicting the miRNA defense regulatory network in the IR56 rice against BPH infestation. The findings from the study add further insights into the molecular mechanisms of rice?BPH interactions and will be helpful for the future researches.

Ethylene response of salt stressed rice seedlings following Ethephon and 1-methylcyclopropene seed priming.

Salinity stress is one of the most devastating abiotic stresses affecting rice growth and productivity. Several phytohormones in rice contribute towards salinity response, including ethylene (ET). Furthermore, priming of rice seedlings with different chemicals has revealed new insights in rice stress physiology. Therefore, the hydroponic culture experiment under completely randomized design (CRD) factorial was conducted to study the effects of seed priming with two contrasting chemicals 1-Methylcyclopropene (1-MCP) and Ethephon with respect to ET production under salinity stress levels 0 (control, CK) and 25 mM NaCl (salinity stress, SS) on cultivar Zhongzheyou 1 (indica) with five replications. Results revealed that the use of 1-MCP and Ethephon improved overall rice seedling germination and growth as compared to the unprimed one under salinity stress. Early germination rate was found to be higher in the Ethephon primed seedlings. However, the late germination rate, overall seedling vigor index (SVI), total plant dry biomass (higher as 37.8 and 42% in 1-MCP-primed seeds, and 28.1 and 21.3% higher in Ethephon-primed seeds than no primed seeds under both salinity levels), other agronomic traits, and SPAD value were found to be higher in the 1-MCP primed seedlings. Further, the production of reactive oxygen species was comparatively higher in the Ethephon primed seedlings than the 1-MCP-primed seedlings. In addition, evaluation of ET levels and expression profiling (qRT-PCR) of some ET signaling genes revealed that Ethephon priming resulted in elevated ET concentration and upregulated ET signaling genes, including OsACS1 and OsEIN2. Overall, our results provide a basic idea of the role of rice seed priming with Ethephon and 1-MCP under salinity condition and their effects on the ET interplay.

Phenotypic and transcriptomic responses of two Nilaparvata lugens populations to the Mudgo rice containing Bph1.

the Bph1 gene was the first reported brown planthopper (BPH, Nilaparvata lugens) resistance gene in Mudgo rice and was widely used as a commercial cultivar for controlling BPH infestations. However, rapid adaptations of BPH on the Mudgo rice resulted in its resistance breakdown and the emergence of virulent BPH populations. Thus, specific BPH populations and rice varieties can serve as good model systems for studying the roles of different bio-compounds and proteins in the insect-plant interactions. Although our understandings have been improved on the complexity of BPH and rice interactions, the underlying molecular mechanisms remain largely unknown. Here we analyzed the feeding performances and the transcriptomic responses of two BPH populations (Mugdo-BPH and TN1-BPH) during compatible (Mudog-BPH feeding on Mudgo rice) and incompatible (TN1-BPH feeding on Mudgo rice) interactions. The electrical penetration graph (EPG) results indicated that the BPH feeding and performances during the incompatible interaction are significantly affected in terms of decreased honeydew, loss of weight, decreased phloem sap ingestion (N4 waveform), but increased non-penetration (NP waveform) phase. Abundance of glucose and trehalose was reduced in BPH during the incompatible interaction. Transcriptomic surveys of insects in both interactions revealed that genes involved in cuticle formation, detoxification, metabolite transport, digestion, RNA processing, lipid or fatty acid metabolism, and proteolysis were significantly down-regulated during the incompatible interaction, whereas genes involved in insulin signaling were significantly upregulated. Knockdown of four genes, including the sugar transporter NlST45, the serine and arginine-rich protein NlSRp54, the cytochrome P450 gene NlCYP6AY1, and the cuticle protein NlCPR70 through RNA-interference revealed thess genes are important for BPH survival. Overall, the results of this study will be helpful for the future researches on BPH virulence shifts.

) iTRAQ-based protein profiling and biochemical analysis of two contrasting rice genotypes revealed their differential responses to salt stress.

Salt stress is one of the key abiotic stresses causing huge productivity losses in rice. In addition, the differential sensitivity to salinity of different rice genotypes during different growth stages is a major issue in mitigating salt stress in rice. Further, information on quantitative proteomics in rice addressing such an issue is scarce. In the present study, an isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative protein quantification was carried out to investigate the salinity-responsive proteins and related biochemical features of two contrasting rice genotypes—Nipponbare (NPBA, japonica) and Liangyoupeijiu (LYP9, indica), at the maximum tillering stage. The rice genotypes were exposed to four levels of salinity: 0 (control; CK), 1.5 (low salt stress; LS), 4.5 (moderate salt stress; MS), and 7.5 g of NaCl/kg dry soil (high salt stress, HS). The iTRAQ protein profiling under different salinity conditions identified a total of 5340 proteins with 1% FDR in both rice genotypes. In LYP9, comparisons of LS, MS, and HS compared with CK revealed the up-regulation of 28, 368, and 491 proteins, respectively. On the other hand, in NPBA, 239 and 337 proteins were differentially upregulated in LS and MS compared with CK, respectively. Functional characterization by KEGG and COG, along with the GO enrichment results, suggests that the differentially expressed proteins are mainly involved in regulation of salt stress responses, oxidation-reduction responses, photosynthesis, and carbohydrate metabolism. Biochemical analysis of the rice genotypes revealed that the Na+ and Cl? uptake from soil to the leaves via the roots was increased with increasing salt stress levels in both rice genotypes. Further, increasing the salinity levels resulted in increased cell membrane injury in both rice cultivars, however more severely in NPBA. Moreover, the rice root activity was found to be higher in LYP9 roots compared with NPBA under salt stress conditions, suggesting the positive role of rice root activity in mitigating salinity. Overall, the results from the study add further insights into the differential proteome dynamics in two contrasting rice genotypes with respect to salt tolerance, and imply the candidature of LYP9 to be a greater salt tolerant genotype over NPBA

Genome-wide identification of the SPL gene family in Dichanthelium oligosanthes.

SQUAMOSA promoter-binding protein-like (SPL) transcription factors play vital roles in various plant physiological processes. Although, the identification of the SPL gene family has been done in C4 grass plants, including rice and maize, the same has not been characterized in the C3 grass species Dichanthelium oligosanthes. In this study, 14 SPL genes were identified in the genome of D. oligosanthes. Gene structure analysis of the identified DoSPLs revealed the similarity and redundancy in their exon/intron organizations. Sequence comparisons within the DoSPLs and along with rice SPLs revealed the putative paralogs and orthologs in D. oligosanthes SPL genes. Phylogenetic analysis clustered the DoSPLs into eight groups along with other plant SPLs. Identification of the conserved SBP motifs in all 14 DoSPLs suggested them to be putative SPLs. In addition, the prediction of sub-cellular localization and associated functions for DoSPLs further supported to be SPL genes. The outcome of this study can serve as a framework for the isolation and functional validation of SPL genes in D. oligosanthes.

Differential responses of OsMPKs in IR56 rice to two BPH populations of different virulence levels

The conserved mitogen-activated protein kinase (MAPK) cascades play vital roles in plant defense responses against pathogens and insects. In the current study, the expression profiles of 17 OsMPKs were determined in the TN1 and IR56 rice varieties under the infestation of brown planthopper (BPH), one of the most destructive hemimetabolous rice pests. The virulent IR56 BPH population (IR56-BPH) and the avirulent TN1 BPH population (TN-BPH) were used to reveal the roles of OsMPKs in the compatible (IR56-BPH infested on the TN1 and IR56 rice varieties, and TN1-BPH infested on the TN1 rice variety) and the incompatible (TN1-BPH infested on the IR56 rice variety) interaction. The statistical analysis revealed that rice variety, BPH population type, and infestation period have significant effects on the transcription of OsMPKs. Out of these genes, five OsMPKs (OsMPK1, OsMPK3, OsMPK7, OsMPK14, and OsMPK16) were found to exhibit upregulated expression only during incompatible interaction. Six OsMPKs (OsMPK4, OsMPK5, OsMPK8, OsMPK9, OsMPK12, and OsMPK13) were associated with both incompatible and compatible interactions. The transcription analysis of salicylic acid, jasmonic acid, and ethylene phytohormone signaling genes revealed their roles during the rice–BPH interactions. The upregulated expression of OsC4H, OsCHS, and OsCHI in the incompatible interaction implied the potential defense regulatory roles of phenylpropanoids. In both varieties, the elevated transcript accumulations of OsGST and OsSOD, and the increased enzyme activities of POD, SOD, and GST at 1 day post-infestation (dpi), but not at 3 dpi, indicated that reactive oxygen species (ROS) signaling might be an early event in rice–BPH interactions. Furthermore, upregulated transcription of OsLecRK3 and OsLecRK4 was found only during an incompatible interaction, suggesting their involvement in the BPH resistance response in the IR56 rice variety. Lastly, based on the findings of this study, we have proposed a model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions, respectively

Roles of E93 and Kr-h1 on metamorphosis of Nilaparvata lugens.

Metamorphosis is a crucial process in insect development. Ecdysone-induced protein 93 (E93) is a determinant that promotes adult metamorphosis in both hemimetabolous and holometabolous insects. Krüppel-homolog 1 (Kr-h1), an early juvenile hormone (JH)- inducible gene, participates in JH signaling pathway controlling insect metamorphosis. In the current study, an E93 cDNA (NlE93) and two Kr-h1 cDNA variants (NlKr-h1-a and NlKr-h1-b) were cloned from Nilaparvata lugens (Stål), one of the most destructive hemimetabolous insect pests on rice. Multiple sequence alignment showed that both NlE93 and NlKr-h1 share high identity with their orthologs from other insects. The expression patterns revealed that decreasing NlKr-h1 mRNA levels were correlated with increasing NlE93 mRNA levels and vice versa. Moreover, RNA interference (RNAi) assays showed that the knockdown of one of the two genes resulted in significantly upregulated expression of the other. Correspondingly, phenotypical observation of the RNAi insects revealed that depletion of NlE93 prevented nymph–adult transition (causing a supernumerary nymphal instar), while depletion of NlKr-h1 triggered precocious formation of incomplete adult features. The results suggest that Nlkr-h1 and NlE93 are mutual repressors, fitting into the MEKRE93 pathway. The balance between these two genes plays a critical role in the metamorphosis of N. lugens determining the proper timing for activating metamorphosis during the nymphal stage

Genetics and molecular mapping of a novel purple blotch resistance gene ApR1 in onion (Allium cepa L.) using STS and SSR markers.

Purple blotch (PB), caused by Alternaria porri (Ellis) Cifferi is the most devastating foliar disease of onion worldwide. However, no attempt has been made so far to detect or map a PB-resistant locus in the onion genome. The present investigation was performed to study the inheritance and develop molecular markers linked to PB resistance by using F1, F2, and BC1 populations developed from a cross between the PB-resistant onion cultivar ‘Arka Kalyan’ and the susceptible parent ‘Agrifound Rose’. Disease evaluation with a virulent isolate of A. porri revealed that the F1 was resistant while 498 F2 plants segregated in a 3:1 resistant (R) to susceptible (S) phenotypic ratio and 128 BC1 lines segregated in 1R:1S ratio, suggesting that the PB resistance is controlled by a single dominant gene designated as ApR1. Out of 288 ISSRs and SSRs primer sets, 59 distinguished the two parental lines and were used in bulk segregant analysis to link them with the presumed ApR1 gene. Seven markers viz. 3 ISSRs (AcISSR471257, AcISSR681600, and AcISSR1031416) and four SSRs (AcSSR7, AcSSR22, AcSSR31, and AcSSR33) showed specific polymorphism between resistant and susceptible bulks and were used for genotyping F2 and BC1 mapping populations. The three ISSR fragments were converted into sequence-tagged markers, and southern blotting confirmed their association with the resistant locus and the single-copy status. Molecular mapping revealed that the SSR marker AcSSR7 and STS marker ApR-450 were closely linked to the ApR1 locus in coupling at distances of 1.3 and 1.1 cM, respectively. Further, both of these markers could not be amplified in 23 susceptible onion genotypes with different genetic backgrounds. This is the first report of identification of markers linked to PBresistant locus in onion. Hence, SSR marker AcSSR7 and STS marker ApR-450 identified in this study could be recommended for facilitating the introgression of ApR1 into susceptible onion variants for the development of high yielding PB-resistant genotypes.

In silico analysis of onion chitinases using transcriptome data.

Chitinases are glycoside hydrolase (GH) family of proteins having multifaceted roles in plants. It is of interest to identify and characterize chitinase-encoding genes from the popular bulbous plant onion (Allium cepa L.). We have used the EST sequences for onion chitinases to elucidate its functional features using sequence, structure and functional analysis. These contigs belong to the GH19 chitinases family according to domain architecture analysis. They have highly conserved chitinase motifs including motifs exclusive to plant chitinases as implied using the MEME based structural characterization. Estimation of biochemical properties suggested that these proteins have features to form stable and hydrophilic proteins capable of localizing extracellular and in vacuoles. Further, they have multiple cellular processes including defense role as inferred by DeepGO function prediction. Phylogenetic analysis grouped them as class I and class VII plant chitinase, with possible abundance of class I chitinase in onion. These observations help in the isolation and functional validation of onion chitinases.

Mining and characterization of Allium cepa expressed sequence tags (ESTs) encoding receptor-like kinases (RLKs).

Plant receptor-like kinases (RLKs) are comprised of a large family of kinases having multitudinous functions. Onion (Allium cepa), an important bulbous plant, has almost no reports on RLKs and their role in onion plant growth and defense responses. In the present work, mining of the transcript database ‘dbEST’ has been performed to obtain the expressed tags of A. cepa. Subsequently, the consensus sequence assembling carried out by CAP3 program resulted in 4175 number of contigs. Consecutive rounds of BLAST searches resulted in identification of 4 highly homologous contigs with the previously reported plant RLKs. Further structural characterizations by MEME, Prosite and SOPMA supported the identified contigs to be putative RLKs. All four putative RLKs (AcCon13, AcCon183, AcCon383, and AcCon1738) possessed 9 out of 11 canonical kinase sub-domains, the signature protein-kinase domain, and the ATP binding sites. Phylogenetic analysis along with previously reported plant RLKs revealed that the identified contigs belong to different RLK sub-families.

Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. cepae.

The basal plate rot fungus, Fusarium oxysporum f. sp. cepae (FOC), is the most devastating pathogen posing a serious threat to garlic (Allium sativum L.) production worldwide. MicroRNAs (miRNAs) are key modulators of gene expression related to development and defense responses in eukaryotes. However, the miRNA species associated with garlic immunity against FOC are yet to be explored. In the present study, a small RNA library developed from FOC infected resistant garlic line was sequenced to identify immune responsive miRNAs. Forty-five miRNAs representing 39 conserved and six novel sequences responsive to FOC were detected. qRT-PCR analyses further classified them into three classes based on their expression patterns in susceptible line CBT-As11 and in the resistant line CBT-As153. North-blot analyses of six selective miRNAs confirmed the qRT-PCR results. Expression studies on a selective set of target genes revealed a negative correlation with the complementary miRNAs. Furthermore, transgenic garlic plant overexpresing miR164a, miR168a and miR393 showed enhanced resistance to FOC, as revealed by decreased fungal growth and up-regulated expression of defense-responsive genes. These results indicate that multiple miRNAs are involved in garlic immunity against FOC and that the overexpression of miR164a, miR168a and miR393 can augment garlic resistance to Fusarium basal rot infection

Development of a Sequence Tagged Site (STS) Marker for sex identification in the dioecious rattan species Calamus guruba Buch.-Ham.

Calamus guruba Buch.-Ham., a perennial, dioecious rattan species of the family Arecaceae has recently emerged as an important source of tribal economy owing to its high-quality flexible canes for furniture manufacturing and cottage industries. The dioecious nature together with disproportionate distribution of male and female sex organs has been the major constraint in the development of an effective breeding programme for the plant. Early identification of the male and female genotypes at the seedling stage is a pre-requisite to ensure genetic improvement of C. guruba. In the present study, we used 30 inter simple sequence repeat (ISSR) markers to develop a sequence-tagged site (STS) linked to male sex in C. guruba. Molecular analysis of bulked DNA pooled from male and female genotypes resulted in the isolation of a putative male-specific marker CgMSM. Partial sequencing of CgMSM resulted in an STS marker CgMY1 which could successfully amplify a 597-bp fragment in male but not in the female plants. DNA gel blot analysis confirmed it as a single-copy locus in the male genome of C. guruba. Further validation of CgMY1 in the natural population of C. guruba resulted in precise detection of 9 males and 6 females from 15 individuals with unknown sex. Therefore, the STS marker CgMY1 could be used as a proficient tool for early sex differentiation and form the basis of a sustainable breeding programme for genetic improvement of C. guruba.

Differential expression of defense-related genes in chili pepper infected with anthracnose pathogen Colletotrichum truncatum

Anthracnose disease caused by Colletotrichum truncatum is a major economic constraint to chilli production in the tropical and subtropical regions of the world including India. To understand the molecular mechanisms of defense against anthracnose, we used a differential chilli-C. truncatum system consisting of two cultivars of chilli (Susceptible; Teja Jhal, TJ and resistant; Bhut Jolokia, BJ) and a highly virulent C. truncatum isolate to evaluate the temporal expression of seventeen defense-related genes associated with multiple defense signaling pathways. Quantitative real time PCR demonstrated rapid induction and significant accumulation of jasmonic acid (JA) and ethylene (ET) responsive genes such as plant defensin 1.2, Lypoxygenase 3, Allene oxide synthase and ACC synthase 2 in the resistant cultivar post treatment with C. truncatum. Significant induction of these genes was also realized upon exogenous treatment with JA and ET. The salicylic acid (SA) responsive phenylalanine ammonia-lyase 2 gene was upregulated only under incompatible interaction. Further, the transcript levels of pathogenesis related proteins PR2 and PR5 was significantly higher in the resistant genotype whereas PR1 and PR3 were moderately responsive. In addition, the expression of defense responsive transcription factors increased gradually in the resistant cultivar and remained significantly higher at 9 dpi as opposed to the susceptible one. Combined result analyses revealed that chilli adopts multiple defense strategies in mediating defence responses against the C. truncatum infection. Overall, this work represents a valuable resource for future functional genomics experiments to unravel the molecular mechanisms of host resistance and pathogen virulence in the agriculturally important chilli-C. truncatum pathosystem

Regulation of miR394 in response to Fusarium oxysporum f. sp. cepae (FOC) infection in garlic (Allium sativum L).

MicroRNAs (miRNAs) are a class of post-transcriptional regulators that negatively regulate gene expression through target mRNA cleavage or translational inhibition and play important roles in plant development and stress response. In the present study, six conserved miRNAs from garlic (Allium sativum L.) were analyzed to identify differentially expressed miRNAs in response to Fusarium oxysporum f. sp. cepae (FOC) infection. Stem-loop RT-PCR revealed that miR394 is significantly induced in garlic seedlings post-treatment with FOC for 72 h. The induction of miR394 expression during FOC infection was restricted to the basal stem plate tissue, the primary site of infection. Garlic miR394 was also upregulated by exogenous application of jasmonic acid. Two putative targets of miR394 encoding F-box domain and cytochrome P450 (CYP450) family proteins were predicted and verified using 5? RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends) assay. Quantitative RT-PCR showed that the transcript levels of the predicted targets were significantly reduced in garlic plants exposed to FOC. When garlic cultivars with variable sensitivity to FOC were exposed to the pathogen, an upregulation of miR394 and down regulation of the targets were observed in both varieties. However, the expression pattern was delayed in the resistant genotypes. These results suggest that miR394 functions in negative modulation of FOC resistance and the difference in timing and levels of expression in variable genotypes could be examined as markers for selection of FOC resistant garlic cultivars.

Identification of novel source of resistance and differential response of Allium genotypes to purple blotch pathogen, Alternaria porri (Ellis) Ciferri.

Purple blotch, caused by Alternaria porri (Ellis) Cifferi, is a serious disease incurring heavy yield losses in the bulb and seed crop of onion and garlic worldwide. There is an immediate need for identification of effective resistance sources for use in host resistance breeding. A total of 43 Allium genotypes were screened for purple blotch resistance under field conditions. Allium cepa accession ‘CBT-Ac77’ and cultivar ‘Arka Kalyan’ were observed to be highly resistant. In vitro inoculation of a selected set of genotypes with A. porri, revealed that 7 days after inoculation was suitable to observe the disease severity. In vitro screening of 43 genotypes for resistance to A. porri revealed two resistant lines. An additional 14 genotypes showed consistent moderate resistance in the field as well as in vitro evaluations. Among the related Allium species, A. schoenoprasum and A. roylei showed the least disease index and can be used for interspecific hybridization with cultivated onion. Differential reaction analysis of three A. porri isolates (Apo-Chiplima, Apn-Nasik, ApgGuntur) in 43 genotypes revealed significant variation among the evaluated Allium species (P = 0.001). All together, the present study suggest that, the newly identified resistance sources can be used as potential donors for ongoing purple blotch resistance breeding program in India

Curcuma longa mitogen-activated protein kinase 6 (ClMPK6) stimulates the defense response pathway and enhances the resistance to necrotrophic fungal infection.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that transduce the externally perceived signals and play crucial role in plant defense against pathogen attack. In the present study, a turmeric (Curcuma longa L.) complementary DNA (cDNA) encoding a MAPK gene responsive to Pythium aphanidermatum infection was isolated using rapid amplification of cDNA ends (RACE)-PCR. It was designated as ClMPK6 based on its high homology with Arabidopsis AtMPK6. The full-length cDNA of 1484 bp length carried an open reading frame (ORF) of 1176 bp encoding a 391 amino acid polypeptide. ClMPK6 protein contains Thr-GluTyr (TEY) motif on its activation loop with a common docking (CD) domain at the C-terminal end and belong to subgroup A of MAPK family. Southern hybridization revealed single copy of ClMPK6 in turmeric genome, and its intron-exon composition showed highly conserved nature of these signaling kinases across different species. Quantitative RT-PCR showed high expression of ClMPK6 in rhizome tissues of mature turmeric plants. Analysis of temporal expression revealed significant induction of ClMPK6 transcript in response to defense signaling molecules and pathogen attack at the early stages. Ectopic overexpression of ClMPK6 in Arabidopsis plants resulted in enhanced resistance to Botrytis cinerea and constitutively high expression of defense responsive genes like PDF1.2, PAD3, AOS, ACS2, ACS6, etc. Our results suggest that ClMPK6 substantiate the characteristics of AtMPK6 orthologs in defense against necrotrophic infection in plants.

Identification and characterization of microRNAs in turmeric (Curcuma longa L.) responsive to infection with the pathogenic fungus Pythium aphanidermatum.

Soft rot fungus, Pythium aphanidermatum, is the most devastating pathogen posing a serious constraint to turmeric (Curcuma longa L.) production worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that act as important modulators of gene expression related to several stress responses. However, to the best of our knowledge, there is no report on miRNA expression profiling in turmeric and miRNA functions in response to P. aphanidermatum remains unclear. In the present study, we sequenced a small RNA library developed from P. aphanidermatum infected turmeric rhizomes and employed a de novo assembled turmeric genome for miRNA prediction and target characterization. In total, 28 miRNAs representing 25 conserved and three novel sequences responsive to P. aphanidermatum were identified. Stem-loop reverse transcription PCR validated the expression of representative miRNAs from different families. Further, we computationally predicted the miRNA targets, many of which were experimentally confirmed using ligation mediated 50 rapid amplification of cDNA ends analysis. Quantitative RT-PCR analysis of 12 selected miRNAs revealed simultaneous reciprocal changes in the expression patterns of six miRNAs (miR159, miR160, miR164, miR393, miR482 and miRx2) and their target genes in the infected rhizomes, thus suggesting their critical involvement in the modulation of a compatible interaction between turmeric and P. aphanidermatum. Our results suggest that P. aphanidermatum may have developed a virulence mechanism by interfering with plant miRNAs like miR164, miR393, miR482 and miRx2 to reprogram the defense gene expression in turmeric and facilitate pathogenesis

De novo sequencing and characterization of defense transcriptome responsive to Pythium aphanidermatum infection in Curcuma longa L.

The rhizome soft rot caused by the necrotrophic oomycete, Pythium aphanidermatum is a serious constraint to turmeric (Curcuma longa L.) production worldwide. The molecular mechanisms underlying the pathological response of turmeric to P. aphanidermatum are not fully understood. In the present study, we performed a deep transcriptome profiling of a resistant turmeric genotype (Cl210-27) post inoculation with P. aphanidermatum. De novo assembly of 146 million clean RNA-Seq reads resulted in 55,014 contigs with length >200 bp. Of these, 34,185 (62.14%) contigs showed significant similarity to the protein sequences from the non-redundant (nr) protein database. 2978 contigs were identified as differentially expressed between infected and mock samples, 2231 of which were up-regulated and 747 were down-regulated. Functional annotation revealed high enrichment of transcripts associated with plant-pathogen interaction, defense signalling and plant secondary metabolism. Rapid induction of pathogen responsive genes encoding NBS-LRRs, glucanases, peroxidases, receptor kinases as well as those associated with jasmonate and ethylene signalling suggest that C. longa adopts multiple strategies to defend against pathogen attack. Further, the identification of multiple miRNA targets confirms the critical involvement of post-transcriptional gene silencing mechanisms in regulating resistance response in C. longa. Overall, the present study represents a comprehensive picture of genes associated with rhizome rot resistance and represents a valuable resource for future function

Molecular characterization and heterologous expression of a pathogen induced PR5 gene from garlic (Allium sativum L.) conferring enhanced resistance to necrotrophic fungi.

Pathogenesis-related protein-5 (PR5) is encoded by a complex group of gene family that are involved in host defense against biotic and abiotic stresses as well as regulation of physiological processes in a wide range of plants and animals. In the present study, we isolated and characterized a PR5 gene, designated as AsPR5, induced in response to Fusarium oxysporum f. sp. cepae (FOC) infection in garlic (Allium sativum). AsPR5 cDNA encoded a protein of 223 amino acids including a 22 amino acid signal peptide at the N-terminus suggesting that it is an apoplast secreted acidic protein. AsPR5 protein contains 16 conserved cysteine residues and five additional conserved amino acids- an arginine, a glutamic acid and three aspartic acids related with antifungal activity of most plant thaumatin like proteins. Semi-quantitative RTPCR showed that the transcript levels of AsPR5 was higher in stem tissues, the primary site of FOC infection, followed by leaves, roots and flowers. Temporal expression analysis using qPCR revealed high expression of AsPR5 upon infection with FOC as well as various phytohormones (JA, ET and ABA) and stress stimuli (wounding, high temperature and salinity) indicating its involvement in both biotic and abiotic stresses. Analyses of purified recombinant protein against different fungal pathogens showed improved antifungal activity compared to other reported PR5 proteins. Furthermore, the Arabidopsis plants ectopically expressing AsPR5 showed enhanced resistance to fungal pathogen, Botrytis cinerea and constitutively higher expression of defense responsive genes such as Lox3, PDF1.2, PAD3, AOS, and AOC. These results suggest that, besides antifungal activities against the necrotrophs, AsPR5 also play significant role in activating multiple defense pathways for enhancing stress resistance in crop plants

Evaluation of cultivated and wild Allium accessions for resistance to Fusarium oxysporum f. sp. cepae.

Fusarium basal rot (FBR) caused by Fusarium oxysporum f. sp. cepae (FOC) is a highly destructive soil borne disease incurring heavy damage in pre and post harvest onion and garlic crops worldwide. Only a few onion lines exhibit partial resistance against the pathogen and there is a need for identification of more effective resistance sources for use in breeding programmes. Selected sets of wild onion and garlic accession and seven related Allium species were screened for resistance to Fusarium basal rot using three FOC isolates. FOC infection revealed significant variation among the evaluated Allium species (at P = 0.001). A. sativum accession ‘CBT-As153’ showed high level of resistance to each isolate while A. cepa accession ‘CBT-Ac77’ exhibited intermediate resistance. Among related Allium species, A. fistulosum, A. roylei and A. schoenoprasum were highly resistant, A. tuberosum had mixed response while A. griffithianum was susceptible. Further, the root density of Allium species negatively correlated with disease incidence for different FOC isolates. Thus, the present study suggests that besides related Allium species, A. sativum ‘CBT-As153’ can be used as a potential donor of FBR resistance for genetic improvement of onion and garlic in India

Mining, characterization and validation of EST derived microsatellites from the transcriptome database of Allium sativum L.

Expressed Sequence Tags (ESTs) with comprehensive transcript information are valuable resources for development of molecular markers as they are derived from conserved genic regions. The present study highlights the mining of EST database to deduce the class I hyper variable SSRs in A. sativum. From 21694 garlic EST sequences, 642 non-redundant SSRs were identified with an average frequency of 1 per 14.9 kb of garlic transcriptome. The most abundant SSR motifs were the mononucleotides (32.86%) followed by trinucleotides (28.50%) and dinucleotides (13.39%). Among the individual SSRs, (A/T)n accounted for the highest number (137; 21.33%) followed by (G/C)n (74; 11.52%) and (AAG)n (63;9.81%). Primers designed from a robust set of 7 AsESTSSRs resulted in the amplification of 63 polymorphic alleles in 14 accessions of garlic. The resolving power of the markers varied from 4.286 (AsSSR7) to 18.143 (AsSSR13) while the average marker index (MI) was 5.087. These EST-SSRs markers for garlic could be useful for the improvement of garlic linkage map and could be used for evaluating genetic variation and comparative genomics studies in Allium species.

Molecular cloning and expression analysis of four turmeric MAP kinase genes in response to abiotic stresses and phytohormones.

Plant mitogen activated protein kinase (MAPK) cascades comprise a complex network playing a major role in regulating extracellular stimuli as well as developmental processes. The present study involves cloning four MAPKs (ClMPK1, 3, 4 and 5) from Curcuma longa. All four ClMPKs have fully canonical motifs of MAPK and each is represented by a single copy in the turmeric genome. The analysis of exon-intron junctions revealed conserved nature of ClMPKs across different plant groups. The RT-qPCR analysis showed their expression in mature plant tissues. The transcript analysis using the RT-qPCR shows that the four ClMPKs were differentially regulated by cold, salinity, and drought stresses. ClMPK4 showed a significant upregulation in the presence of NaCl, polyethylene glycol, and mannitol.

An APETALA3 MADS-box linked SCAR marker associated with male specific sex expression in Coccinia grandis (L). Voigt.

Coccinia grandis L. (Family – Cucurbitaceae), a perennial dioecious herb with heteromorphic sex chromosomes has the quality of a model plant for analysis of sexual evolution in angiosperms. Yet, little is known about the physical side, genetic orientation and substitution behavior of key gender-defining factors in this important plant species. Screening of genomic DNA with RAPD primers was used for sex diagnosis and gender specificity of C. grandis in this study. Bulk analyses of pooled DNA from male and female genotypes resulted in the isolation of a putative male specific sex marker CgMSM. A sequence characterized amplified region (SCAR) marker CgY1 designed from CgMSM amplified an 829 bp fragment in the male C. grandis but not in the female plants. Southern blotting confirmed it as a single copy locus in the male genome.

Development of an ISSR based STS marker for sex identification in pointed gourd (Trichosanthes dioica Roxb.).

Pointed gourd (Trichosanthes dioica Roxb.), a perennial dioecious plant of the family Cucurbitaceae has recently emerged as an important cash crop due to its great medicinal and nutritive value. Breeding programmes in pointed gourd have several constraints such as poor germination, vegetative means of propagation and dioecy. Identification of male and female at the seedling stage is a pre-requisite for the genetic improvement of this species. Screening of genomic DNA with inter simple sequence repeat (ISSR) primers was used for early detection of male and female plants in this study. Using pooled DNA from male and female genotypes and 40 ISSR primers, a putative male specific marker TdMSM was produced. It was converted into a sequence tagged marker TdSTSM which could successfully amplify 720 bp product in male but not in the female.

Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum.

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs ?-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with ?-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved

Molecular characterization of NBS encoding resistance genes and induction analysis of a putative candidate gene linked to Fusarium basal rot resistance in Allium sativum.

Fusarium basal plate rot (FBR), caused by Fusarium oxysporum f. sp. cepae (FOC), is one of the most devasting soil-borne disease of onion and garlic (Allium sativum) worldwide. Resistance screening to FBR has resulted in identifying a garlic selection line CBT-As153 as resistant to FOC. Majority of plant disease resistance (R) genes encode a highly conserved nucleotide binding site and leucine-rich repeat structure (NBS–LRR) which can aid to isolate candidate genes linked to FBR resistance in CBT-As153. Degenerative primers based on the NBS conserved motif of NBS–LRR resistance proteins isolated 28 NBS sequences from CBT-As153 named as A. sativum resistance gene analogs (AsRGAs). Sequence analysis grouped the AsRGAs into six classes of non-toll interleukin receptor (non-TIR) subfamily. Quantitative real time polymerase chain reaction (qRT-PCR) revealed differential transcript expression

Molecular characterization and functional analysis of CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum.

Rhizome rot disease caused by necrotrophic oomycete Pythium aphanidermatum is responsible for upto 60% of yield losses in turmeric (Curcuma longa L). However, Curcuma zedoaria L, a wild relative of turmeric, is resistant to P. aphanidermatum and has been proposed as a potential donor for rot resistance to C. longa. We used a previously isolated resistance gene candidate Czp11 from C. zedoaria as a template to characterize a major resistance gene CzR1 through candidate gene approach in combination with RACE-PCR strategy. CzR1 encodes a 906 amino acid predicted protein with a calculated pI of 8.55. Structural and phylogenetic analyses grouped CzR1 within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS-LRR R-genes. Reverse transcription PCR revealed specific transcript expression of CzR1 only in P. aphanidermatum resistant lines of C. zedoaria and Zingiber zerumbet

Rice Production Under Climate Change: Adaptations and Mitigating Strategies.

In the current scenario of global climate change, the utmost desire to ensure food security is to maintain and increase agricultural production. But, due to rapid climate change, many abiotic factors such as rainfall, drought, flooding, temperature and solar radiations are severely affecting the production of rice at various growth stages. It is predicted that almost 51% of rice cultivation and production would be reduced during the next century due to global climate change. However, agriculture activities are also contributing to global warming by 10–14% of total global greenhouse gas emissions and 18% of the total methane is emitted from paddy rice fields. Therefore, mitigating and adaptation strategies such as alternate wetting and drying, inter cropping with short term vegetation, limiting chemical fertilizers by precise farming, usage of rice cultivars with low methane emission, improved tillage, recycling of farm

Endophyte mediated host stress tolerance as a means for crop improvement.

Plants being sessile are continuously exposed to a wide range of biotic and abiotic stresses that exert adverse effect in their growth and development. Various physiological, biochemical, and molecular machineries are employed by the plants to overcome these stresses. Endophytes are mostly the symbiotic fungi and bacteria that reside inside the plant tissue and stimulate plant growth during stress conditions. Endophyte-mediated plant stress tolerance holds significant role in the analysis of plant-microbe interactions. Although still at its infancy, the endophyte-mediated host stress tolerance including drought, salinity, high-temperature stresses, and pathogenic infection has been well described in the recent times. The molecular mechanism governing the endophyte-mediated stress response includes the induction of plant stress genes and regulation of reactive oxygen species. In the present review, we discuss the evidences for bacterial and fungal endophyte-mediated stress tolerance and associated mechanisms. This information from this review will help the scientific community in the development of suitable biotechnological approaches toward usage of endophyte microbes in the improvement of crop yield under multiple stress conditions

The CRISPR/Cas Genome editing system and its application in rice improvement.

Rice (Oryza sativa L.) is the major staple food crop in the world, feeding more than 50% of the world’s population (Verma et al., 2012, 2013, 2015). Rapid increase in the human population together with acute weather conditions leading to biotic and abiotic stresses demand for a sustainable improvement of crop varieties including rice. Conventional rice improvement methods such as cross hybridization and mutation breeding techniques are gradually getting inhibited by the declination of the existing genetic variability thereby affecting the production for future supply (Chen and Gao, 2014). Molecular marker-assisted selection has the advantage of detecting constructive agricultural characters. However, large dependence on traditional crossing and backcrossing approaches for pyramiding of agriculturally important traits makes the process both times consuming and labor-intensive for rice improvement.

Metabolic engineering of phenylpropanoids in plants.

Phenylpropanoids are diverse group of active secondary metabolites derived from the carbon backbone of amino acids phenylalanine and tyrosine. The phenylpropanoid pathway serves as the starting point for the biosynthesis of a wide range of organic compounds such as lignins, flavanols, isoflavanoids, anthocyanins, and stilbenes with an array of important functions including plant defense and structural support. Besides, they have major nutritional and pharmaceutical properties that find uses as food supplements, antioxidants, flavoring agents, insecticides, dyes, and pharmacological drugs. Major structural and regulatory genes of the phenylpropanoid pathway and associated branches have been isolated and characterized in the recent times.

From Phytochemicals to Phytomedicines: Potential Roles of Plant-Based Biomolecules in the Covid Era

The current pandemic of the Corona virus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has urged for the invention and implementation of effective drugs and vaccines to mitigate the adversity worldwide. Numerable studies are going on to identify and evaluate the efficacy of several synthetic drugs and vaccines. In this scenario, identification and use of plant-derived biomolecules against SARS-CoV-2 could be highly beneficial. Furthermore, upscale production of such plant metabolites and plant-based vaccines can help in controlling the pandemic. Several previous studies have reported the success of plant-based traditional medicines in immunity enhancement and decreasing viral loads. Thus, in depth researches involving the phytochemicals could reveal their roles and level of efficacy against SARS-CoV-2. Considering the present scenario, this review article presents the perspectives of using the phytochemicals in mitigating SARS-CoV-2, and the possible evolution of these phytochemicals into phytomedicines.

Molecular cytogenetics in agriculture: A perspective on rice improvements

Molecular basis of insect resistance in plants: Current updates and future prospects

Insect pests are one of the major threats to plant productivity and survival. Plants respond to insect attack through perception of insect effectors and activation of multiple signaling components including reactive oxygen species, Ca2+ and MAP kinases. The defense signaling in turn is regulated by phytohormones, secondary metabolites, volatile compounds, physical barriers, defense proteins and transcription factors. The emergence of genomics and genetic engineering has facilitated the identification of multiple molecular components and their usage in inducing insect resistance. In the present review, we discuss about various strategies used by plants in response to insect herbivory with special reference to various molecular and genomic approaches towards insect resistance. Expansion of molecular approaches to understand plant-insect interaction will be a priority in future towards development of novel insect resistant plant varieties.

Molecular perspectives of plant-pathogen interactions: An overview on plant immunity.

In nature, there is a constant arm race is going on in between plants and pathogens. These plant-pathogen interactions are complex and multifaceted. To tackle the invading pathogens, plants have developed multiple resistance responses at several levels. On the contrary, adapting capabilities and evolution of new effector molecules help the phytopathogens to outrun plant defenses and proliferate in the host cells. Although, many theories and models have been proposed to address these interactions, none of them are exhaustive and fully understood. Therefore, it is essential to make a comprehensive summery of the existing plant-pathogen interaction models and delineate their intricacy related to plant protection. In this review, two crucial pathways of plant immune response, including the pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) has been discussed elaborately. As both PTI and ETI are the major pathways involved in plant immunity, understanding their details and the key players involved in these cascades will be highly beneficial. In addition, a comparative discussion of the different models proposed for understanding the ETI has been presented. Understanding of these complex interactions can facilitate the unravelling of the involvement of different plant resistance pathways. Moreover, the review will serve as a basic layout to have an overview of the molecular mechanisms of plant immune responses against phytopathogens.

Utilization of seaweed-based biostimulants in improving plant and soil health: Current updates and future prospective

Seaweeds are valuable marine resources having multifaceted usages in modern agriculture. Extracts from different seaweeds have been used since many years in agriculture practice to improve plant growth and to enhance the yield. At present, the excessive use of synthetic chemicals and fertilizers imposes a global health hazard and pollutes agricultural lands worldwide. Thus, promoting the use of natural plant growth regulators and fertilizers, such as seaweeds, is recommended with promising outputs. Moreover, the seaweed extract-based biostimulants having plant growth -promoting capacity and aiding in improving the overall plant resilience are highly desirable. In addition, the seaweed extracts can improve the soil health and soil fertility. In this review, the different methods of obtaining the seaweed extract-based biostimulants are discussed. Additionally, the role of such biostimulants in plant growth promotion, mitigating plant abiotic and biotic stresses, and improving soil quality is illustrated and the underlying mechanism is discussed. In addition, some important applications of the seaweed extracts in other than agricultural area have been discussed in brief. Moreover, the future prospects of the seaweed extract-based biostimulants are presented with emphasizing

Cultivar Mixture: Old but Unique Part of Integrated Disease Management

The two most commonly used disease management methods prevalent in agricultural package of practices are: the application of chemicals and the selection of disease-resistant cultivars through the introduction of resistance genes. Each of these preventive measures are equally susceptible to the adaptations of phytopathogens in due course of time. There are numerous records of several phytopathogens overcomimg the resistance provided by the main resistance gene through continuous evolution. In a similar fashion, the development and fixation of pesticides resistance mutations in the phytopathogens has made many pesticides to lose their effectiveness. The existingdisease management practices are presumably deficient in supporting the sustainable intensification of crop productivity due to its inadequacy of minimizing the phytopathogen evolution. Thus, comes the role of cultivar mixture which is based on the principle of supressing the evolution of phytopathogen against a specific gene or pesticide. Combinations of cultivars having several different characteristics but sufficient morphological and physiological similarities can be cultivated collectively. These cultivar mixtures do not cause any significant changes in crop production system, yet improve the yield accuracy, and decrease the use of pesticides in many cases. They are also faster and cheaper to develop and are distinctive to “multiline” which are identified as combinations of genetically identical lines of a crop species varying mostly in disease resistance gene. The use of cultivar mixtures can improve the effectiveness of disease management practices as their level of resistance

Biocontrol of Fusarium Wilt in Tomato: An Eco-friendly and Cost Effective Approach

Tomato is the second most widely produced vegetable in the world. All through growth time or even after harvesting, tomatoes are prone to several diseases brought about through viruses, fungal pathogen, bacteria, and nematodes. Tomato wilt is a fungal disease, caused by Fusarium oxysporum f.sp. lycopersici, that limiting tomato output severely around the world. Several measures were offered to restrict the spread of Fusarium oxysporum f.sp. lycopersici but were difficult because of the ability of the fungus to stay long in the soil. A wide range of chemical pesticides currently available and the continuous use of these pesticides affect the food substance of tomatoes as well as their texture and the performance of soil in order to control all these vulnerabilities. Thus, this review focuses on the possible use of microbial pesticide potential along with mechanisms for the management of wilt disease in tomato. Additionally, different constraints in tomato production, symptoms produced by wilt disease, nature of causal organism, epidemiology and losses, infection stages and disease cycle are also discussed. The use of a microbial pesticide is an environmentally friendly and effective way to prevent tomatoes from wilt disease and its devastating consequences. Different microorganisms have been used in tomato wilt treatment and are now being discussed. The beneficial inoculum not only suppresses the disease, but it also contributes in the healthy growth of crops.

Beneficial Microorganisms for Achieving Stable and Sustainable Agriculture

There is immense pressure on farmers to increase crop production for which they injudiciously use synthetic fertilizers and pesticides without thinking about their adverse environmental impact. Apart from affecting the health of the environment and humans, the increased application of synthetic fertilizers also lowers the profit due to their cost. As a result, an agricultural production approach that is environmentally friendly, cost effective, and can increase yield and profitability simultaneously is required. The use of beneficial microbes helps both crop and the soil through a variety of complex methods such as biological fixation of nitrogen, systematic resistance development, antibiotic synthesis, siderophores, organic acids, growth hormones, enzymes, and many others. Some of the commonly used beneficial microorganisms in agriculture belong to the genus Rhizobium, Azospirillum, Bacillus, Pseudomonas, Trichoderma, Cyanobacteria, Streptomyces, Mycorrhizae, etc. The incorporation of these beneficial microbes with modern production techniques can certainly help in improving agricultural production worldwide. This article discusses in brief the benefits of these microorganisms in agricultural production and the mechanisms underlying those benefits.

Genome-wide Identification of the GATA Transcription Factor Family in Dichanthelium oligosanthes.

The GATA transcription factors (TFs) play a crucial role in regulating various physiological processes in plants. Identification and characterization of the GATA TF family has been carried out in several important grass species, including rice, maize, and bamboo. However, no information is available on the GATA TFs in the C3 grass species Dichanthelium oligosanthes. In the current study, 31 GATA genes have been identified in the D. oligosanthes genome by stringent bioinformatics analysis. The exonintron arrangement analysis of the DoGATAs via the Gene Structure Display Server (GSDS 2.0) revealed the redundancy and differences in their gene structural organization. In addition, the sequence comparisons within the DoGATAs via BLAST revealed 11 numbers of putative paralogs. Similarly, the BLAST comparisons among the OsGATAs and DoGATAs resulted in the identification of 21 orthologs. Structural analysis of the identified DoGATAs through Simple Modular Architecture Research Tool (SMART), Conserved Domain Database (CDD), and Multiple Expectation Maximization for Motif Elicitation (MEME) revealed that all of them possess the signature GATA domain and the C-X2-C-X18- C-X2-C consensus sequence. The phylogenetic analysis via MEGA divided the DoGATAs into four groups along with rice and Arabidopsis GATAs. In addition, the subcellular localization, gene ontology, and other peptide functional prediction results further supported the DoGATAs to be putative GATA genes. Moreover, the findings of this study can serve as a basic framework for the isolation and functional characterization of GATA genes in D. oligosanthes.

The Auxin and Cytokinin Interplay during Leaf Morphogenesis

Auxins (IAA) and cytokinins (CKs) are the most influential phytohormones, having multifaceted roles in plants. They are key regulators of plant growth and developmental processes. Additionally, their interplay exerts tight control on plant development and differentiation. Although several reviews have been published detailing the auxin-cytokinin interplay in controlling root growth and differentiation, their roles in the shoot, particularly in leaf morphogenesis are largely unexplored. Recent reports have provided new insights on the roles of these two hormones and their interplay on leaf growth and development. In this review, we focus on the effect of auxins, CKs, and their interactions in regulating leaf morphogenesis. Additionally, the regulatory effects of the auxins and CKs interplay on the phyllotaxy of plants are discussed.

RNA Interference: A Functional Genomics Approach for Plant Disease Management

RNA interference machinery is most widely explored by biologist for its conserved mechanism triggered by double stranded RNA (dsRNA), results in silencing of specific genes. It is a process in which small RNAs sequence of 21–30 nucleotides are produced, that regulates the gene expression in a sequence-specific manner. It is a valuable tool for functional genomic studies. Crop plants are subjected to several plant pathogens viz. fungi, bacteria and viruses which not only affect the growth and development of plants but also results in huge yield loss. Thus, plant breeders have adopted various methods to engineer disease resistant plants for effective management of plant diseases. Among them, RNA silencing or RNA interference is used as a powerful tool during past two decades for engineering disease resistant crops. Host-induced gene silencing is mostly used for preventing the plant from disease by silencing the target genes of invading pathogen. Another RNAi-based gene silencing known as (SIGS) spray-induced gene silencing is an innovative disease control strategy used successfully for dicots and monocots against disease causing fungus and past. The present review updates the RNAi technologies and discusses the different strategies that can be adapted towards plant disease management in an eco-friendly manner

Microbe-Assisted Alleviation of Heavy Metal Toxicity in Plants: A Review

Heavy metals contaminations in soil adversely affect the growth and survibality of the plants. Alternatively, plants have evolved complex physiological processes to withstand such environmental cues. Myraids of plant varieties with improved tolerance to metal toxicity has been developed through molecular breeding and transgenic approaches. However, such methods are laborious, expensive and not 100% effective. Recent evidences indicate that several microbes can tolerate metal toxicity and can acclimatize to adverse environmental conditions. The association of plants and such metal-tolerant microbes could be one of the promising strategies to enhance plant performance under metal toxicity. In this review, we focus on the association between plant and microbes in alleviating metal toxicity. We have also presented the different physiological processes of achieving metal toxicity in plants. Furthermore, different strategies to improve plant resilience through microbial applications have been discussed.

Role of molecular markers in improving abiotic stress tolerance in agricultural crops

Molecular markers are one of the expandable inventions in agricultural science. After decades of their introduction, these still are an integral part of crop genotyping and crop improvement. The present scenario offers to choose from different molecular marker types based on their principle, methodologies and extent of applications. Several markers have been used to identify and map abiotic stress related genes and quantitative trait loci (QTL) onto the chromosomes of different crops. The genetic mapping of these identified genetic elements by using different markers provides the scope of selective breeding in the agricultural crops to tackle abiotic stresses. Moreover, recent progress achieved in genetics, molecular breeding and genomic selection has facilitated a comprehensive understanding of the molecular markers and unlocked new insights into marker-assisted breeding. The advent of new technologies like association mapping (AM) and genome-wide association studies has unveiled the highthroughput application of molecular markers. In this review, a focused representation of the use of different molecular markers in the identification of tolerant crop genotypes and associated genes/QTLs against major abiotic stresses, including salinity, drought and temperature has been provided. Additionally, the application of molecular markers in AM and AM-based breeding of some important agricultural crops has been discussed

Omics-assisted understanding of BPH resistance in rice: current updates and future prospective

Rice is one of the most important crops in the world and its production is severely challenged by the brown planthopper (BPH) infestations. Rice and BPH have coevolved and therefore exhibit complex molecular interaction mechanisms. However, the recent advancements in the area of omics, including genomics, transcriptomics, proteomics, and metabolomics huge number of data are being generated with respect to the rice–BPH interactions. These primary data offer a great scope to analyze and draw inferences on the rice immune responses against BPH attacks. In addition, the outcomes of these omics-assisted studies have accelerated the molecular breeding of resistant rice. Further, the transcriptional and proteomic investigations provided deeper insights into the involvement of the different genes and proteins in regulating rice resistance against BPH. Similarly, metabolomic studies have identified the key metabolites in rice–BPH interactions and suggested the different pathways that play a pivotal role in rice resistance. In this chapter a comprehensive representation of the role of genomics, transcriptomics, proteomics, and metabolomics in regulating rice resistance against BPH infestations has been made. In addition, the crucial players involved in each omics during the rice–BPH interactions are discussed extensively. Moreover, this chapter will serve as a brief comprehension to understand the different molecular aspects of the complex rice and BPH interactions.

Oral RNAi toxicity assay suggests clathrin heavy chain as a promising molecular target for controlling the 28-spotted potato ladybird, Henosepilachna vigintioctopunctata

BACKGROUND: Use of RNA interference (RNAi) technology in effective pest management has been explored for decades. Henosepilachna vigintioctopunctata is a major solanaceous crop pest in Asia. In this study, the effects of the RNAi-mediated silencing of clathrin heavy chain in H. vigintioctopunctata were investigated. RESULTS: Feeding either the in vitro-synthesized or the bacterially expressed double-stranded RNAs (dsRNAs) significantly impaired the normal physiology of H. vigintioctopunctata instars and adults. However, the bacterially expressed dsHvChc caused higher mortality than the in vitro-synthesized ones in the larvae and adults. Moreover, on evaluating the potential risk of dsHvChc on Propylea japonica, significant transcriptional effects of dsHvChc1 were observed, while the organismal level effects were not significant. On the contrary, dsHvChc2 did not affect P. japonica at either level. A similar test revealed significant transcriptional effects of dsPjChc1 on H. vigintioctopunctata, while staying ineffective at the organismal levels. Conversely, dsPjChc2 did not affect H. vigintioctopunctata at either level. Importantly, no effect of dsPjChc1 exposure on H. vigintioctopunctata suggested that other factors besides the 21-nucleotide (nt) matches between sequences were responsible. Finally, ingestion of dsHvmChc1 derived from H. vigintioctomaculata, containing 265-nt matches with dsHvChc1, caused 100% mortality in H. vigintioctopunctata. CONCLUSIONS: We conclude that (i) species with numerous 21-nt matches in homologous genes are more likely to be susceptible to dsRNA; (ii) dsRNA can be safely designed to avoid negative effects on non-target organisms at both transcriptional and organismal levels; (iii) HvChc can be used as an efficient RNAi target gene to effectively manage H. vigintioctopunctata.

RNAi suppression of the nuclear receptor FTZ-F1 impaired ecdysis, pupation, and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Fushi-tarazu factor 1 (FTZ–F1) is an ecdysone-inducible transcription factor that plays a vital role during the metamorphosis in insects. In this study, we functionally characterized HvFTZ-F1 in H. vigintioctopunctata, a dreadful solanaceous crop pest, by using a dietary RNA interference technique. The HvFTZ-F1 expression levels were elevated in the 1st and 2nd-instars before molting and declined immediately after ecdysis. The HvFTZ-F1 silencing led to high mortality in the 1st instars, while the expression of the osmosis-regulative gene, HvAQPAn. G, was significantly increased in the 1st instars. HvFTZ-F1 silencing downregulated the Halloween and 20Erelated genes, decreased the ecdysteroids titer, suppressed the expression of pigmentation-related genes, and reduced the catecholamines titer. In the 4th instars, HvFTZ-F1 silencing caused 100% mortality by arresting the development at the prepupal stage and preventing new abdominal cuticle formation. In the female adults, HvFTZF1 silencing caused an evident decrease in fecundity, prolonged the pre-oviposition period, reduced the number of eggs and hatching rate, severely atrophied the ovaries. Moreover, the 20E-related genes and the dopamine synthesis genes were suppressed in the dsHvFTZ-F1-treated females. Overall, our results revealed that HvFTZ-F1 regulates ecdysis, pupation, and reproduction in H. vigintioctopunctata, thereby could be a promising molecular target for the development of RNAi-based biopesticides to control H. vigintioctopunctata.

Genome-wide identification and expression analysis of the RcYABBYs reveals their potential functions in rose (Rosa chinensis Jacq.)

The YABBY transcription factors are evolutionarily conserved in plants . The YABBY genes have been reported in several plants to be involved in vital processes, including growth, development, and stress response. However, no information is available on the rose (Rosa chinensis Jacq.) YABBY gene family. In this study, six rose YABBY genes (RcYABBYs) have been identified . A phylogenetic analysis clustered the identified RcYABBYs into five different sub-families (YAB1/YAB3, YAB2, YAB5, INO, and CRC). Further, structural characterisations revealed that the RcYABBYs possessed the YABBY domains and conserved motifs. The gene ontology (GO) analysis suggested their putative roles in modulating important metabolic pathways in roses, whereas the cis-regulatory element study indicated that the RcYABBYs possess several types of phytohormone and stress-responsive elements. The expression analysis of the RcYABBYs under the exogenous auxin, jasmonic acid, and melatonin applications suggested that the RcYABBYs can be induced by hormonal treatments, while their interactions could be specific to a particular hormone. Similarly, differential expression patterns of the RcYABBYs under the chitosan treatment and Botrytis cinerea infection suggested their possible role in defence regulation in roses. Moreover, the finding of the study added new insights on the structural and functional parts of the YABBY gene family in rose

Tyrosine hydroxylase is involved in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata

BACKGROUND: Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. RESULTS: The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval–larval–pupal cuticle tanning during the third–fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval–pupal–adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. CONCLUSIONS: The collective results of the study suggested that HvTH plays conserved roles in larval–pupal–adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction.

Control efficiency and mechanism of spinetoram seed-pelleting against the striped flea beetle Phyllotreta striolata

The striped flea beetle (SFB, Phyllotreta striolata) can cause serious harm to cruciferous crops in both the larval and adult stages. Presently, there are no other sustainable alternatives to the use of chemical pesticides for controlling SFB infestation. In this study, the use of a seed-pelletized coating of spinetoram effectively reduced the numbers of SFB and its feedings on the flowering cabbage seedlings, whereas, in combination with the insect-proof net, it controlled the SFB infestation throughout the cabbage growth period. The analysis of the pesticide residues in soil and different cabbage parts indicated the degradation dynamics of spinetoram. The concentration of spinetoram in cabbage parts decreased over time, while increased first and subsequently decreased in soil. Furthermore, estimation of the half-life of spinetoram revealed that via seed-palletized application spinetoram half-life was found to be 2.82 days in soil, 4.21 days in the root, 5.77 days in the stem, and 3.57 days in the leaf, respectively. Both the lower pesticide residues and the half-life of spinetoram in soil and cabbage parts suggested it to be a promising environment and food-safe pesticide in controlling SFB. Moreover, the seed-pelletized coating ensured a sustainable release of spinetoram that can reduce the pesticide application frequency and be cost-effective and pocket-friendly for the farmers.

A survey on the off-target effects of insecticidal double-stranded RNA targeting Hv?’COPI gene in the crop pest Henosepilachna vigintioctopunctata through RNA-Seq.

The specificity of the double-stranded RNA (dsRNA) used in the RNA interference (RNAi) technique is crucial for the success of sequence-specific gene silencing. Currently, RNAi-mediated insect control is a trending research topic. However, the off-target effects of the dsRNA in RNAi are a major concern. In this study, the dsHv?´COPI (coat protein complex I, ?´ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle (Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects. The RNA-seq results revealed that 63 and 44 differentially expressed genes (DEGs) were upregulated and downregulated, respectively, in the dsHv?´COPI treated group as compared with the control. Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results. Further downstream analysis revealed that there were no genes homologous with Hv?´COPI in H. vigintioctopunctata. Additionally, no genes with a >11 bp continuous match with dsHv?´COPI were found in the H. vigintioctopunctata transcriptome. Six genes (Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and HvE3 ubiquitin) were selected to examine the off-target activity of dsHv?´COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways. The severity of silencing these six off-target genes was evaluated by employing RNAi. The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality. The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.

Genome-wide identification and expression analysis of CRK gene family in chili pepper (Capsicum annuum L.) in response to Colletotrichum truncatum infection.

The cysteine-rich receptor-like kinases (CRKs) belong to the plant receptor-like kinases family and have multitudinous roles, including stress responses. However, information on CRKs in chili pepper (Capsicum annuum L.) plants is limited. In this study, a total of 22 C. annuum CRKs (CaCRKs) have been identified in the chili genome. All the identified CaCRKs were mapped onto different chili chromosomes. The gene duplication analysis revealed that five gene pairs had the Ka/Ks ratio greater than one, suggesting the positive selection of CaCRKs in chili. Phylogenetic analysis grouped the CaCRKs into four groups alongside other plant CRKs. The structural characterization of the CaCRKs revealed that all of them carry the signature domains and motifs. The gene ontology analysis predicted their major potential functions to be stress response and protein kinase activity. The expression analysis the CaCRKs in two contrasting chili varieties under Colletotrichum truncatum infection added valuable insights into their involvement in chili-C. truncatum interactions. The comparison of expression dynamics suggested that CaCRK13 could be a potential candidate gene in regulating chili defense against anthracnose pathogen. Moreover, the results of this study will help to get insights into the chili CRKs and their involvement in chili defense response against C. truncatum.

Host-delivered-RNAi-mediated resistance in bananas against biotic stresses.

Both the biotic and abiotic stressors restrict the yield potential of many crops, including bananas. Bananas belong to the genus Musa and are the world’s most popular and widely produced fruit for their nutritional and industrial importance. The demand for bananas is growing each day worldwide. However, different pest infestations are hampering the production of bananas, making it a matter of concern for global food security. Several biotechnological tools and applications including RNA interference (RNAi) have been employed to enhance the biotic stress resistance in plants. The capacity to silence targeted genes at transcriptional and post-transcriptional levels makes the RNAi technique a popular choice for gene knock-down and functional genomics studies in crops. Silencing of different suppressor molecule coding genes through RNAi helps crops to combat the detrimental effects of plant pathogens. The host-induced gene silencing (HIGS) technology, also known as the host-delivered RNAi (HD-RNAi), is nowadays gaining popularity due to its ability to target an array of pathogens, comprising bacteria, nematodes, fungi, viruses, and insects. This methodology is employed to manage disease pest outbreaks in a diverse range of crop species, including bananas. Besides HIGS, virus-induced and spray-induced gene silencing (VIGS and SIGS, respectively) are the potential approaches where RNAi technology is exploited to control plant-pathogenic diseases. The current review emphasizes the different kinds of diseases of bananas and the potential of HD-RNAi, a new-age and promising technology to build a barrier against significant crop and economic loss.

Oral RNAi assays in Henosepilachna vigintioctopunctata suggest HvSec23 and HvSar1 as promising molecular targets for pest control

The coat protein II (COPII) complex is a class of coated vesicles involved in protein transport from the endoplasmic
reticulum to the Golgi apparatus, serving a variety of important roles in animal growth and development. In this study,
we selected three COPII genes, HvSec23, HvSec24, and HvSar1 to explore the RNAi silencing effect on Henosepilachna
vigintioctopunctata (HVig), a dreadful solanaceous crop pest, using the dietary RNA interference (RNAi) technique. The
results showed that HvSec23, HvSec24, and HvSar1 had a significant lethal effect on the 1st and 3rd instars of HVig. In addition,
oral administration of the bacterially expressed dsHvSec23 and dsHvSar1 caused high mortality and feeding inhibition
in both larvae and adults, whereas dsHvSec24 only had a significant effect on the larvae. Moreover, the predatory ladybeetle
Propylaea japonica was tested for evaluating the non-target effects of dsHvSec23 through oral and injection RNAi. Results
confirmed that gene expression of PjSec23 was significantly suppressed by injection RNAi, but not oral RNAi. Overall, our
results revealed that HvSec23 and HvSar1 could be used as promising molecular targets for controlling HVig.

Molecular Characterization of bHLH Transcription Factor Family in Rose (Rosa chinensis Jacq.) under Botrytis cinerea Infection

Rose (Rosa chinensis Jacq.) is an important economic ornamental crop and its yield is affected by different biotic and abiotic stresses. Among the biotic stresses, the gray mold disease caused by Botrytis cinerea is a serious threat to rose production. The basic helix-loop-helix (bHLH) is a large transcription factor family involved in several vital plant physiological processes, including growth, development, and stress response. However, no substantial reports exist on bHLH genes in rose. Here, the genome-wide identification, characterization, and expression analysis of the rose bHLH (RcbHLH) genes was carried out. In total, 100 RcbHLHs were identified in the rose genome and mapped onto different rose chromosomes. The gene duplication analysis revealed both tandem and segmental duplications in RcbHLHs. The RcbHLHs among other plant bHLHs were divided into 21 sub-groups on the phylogenetic tree. Additionally, prediction of the different cis-regulatory elements and the gene ontology of the identified RcbHLHs indicated their possible functions in rose plants. The expression analysis of selected RcbHLHs genes in two contrasting rose varieties (A29 = Black Baccara and XS = Sweet Avalanche) under B. cinerea infection provided insights into the involvement of RcbHLHs in rose–B. cinerea interactions. Moreover, this study provided details on the bHLH family genes in rose and their potential roles in rose defense against B. cinerea infection.

Interplay Impact of Exogenous Application of Abscisic Acid (ABA) and Brassinosteroids (BRs) in Rice Growth, Physiology, and Resistance under Sodium Chloride Stress.

The hormonal imbalances, including abscisic acid (ABA) and brassinosteroid (BR) levels, caused by salinity constitute a key factor in hindering spikelet development in rice and in reducing rice yield. However, the effects of ABA and BRs on spikelet development in plants subjected to salinity stress have been explored to only a limited extent. In this research, the effect of ABA and BRs on rice growth characteristics and the development of spikelets under different salinity levels were investigated. The rice seedlings were subjected to three different salt stress levels: 0.0875 dS m?1 (Control, CK), low salt stress (1.878 dS m?1, LS), and heavy salt stress (4.09 dS m?1, HS). Additionally, independent (ABA or BR) and combined (ABA+BR) exogenous treatments of ABA (at 0 and 25 ?M concentration) and BR (at 0 and 5 ?M concentration) onto the rice seedlings were performed. The results showed that the exogenous application of ABA, BRs, and ABA+BRs triggered changes in physiological and agronomic characteristics, including photosynthesis rate (Pn), SPAD value, pollen viability, 1000-grain weight (g), and rice grain yield per plant. In addition, spikelet sterility under different salt stress levels (CK, LS, and HS) was decreased significantly through the use of both the single phytohormone and the cocktail, as compared to the controls. The outcome of this study reveals new insights about rice spikelet development in plants subjected to salt stress and the effects on this of ABA and BR. Additionally, it provides information on the use of plant hormones to improve rice yield under salt stress and on the enhancement of effective utilization of salt-affected soils.

Oral delivery of dsHvUSP is a promising method for Henosepilachna vigintioctopunctata control with no adverse effect on the non-target insect Propylea japonica.

RNAi-based biopesticides against 28-spotted ladybeetle, Henosepilachna vigintioctopunctata does not harm the insect predator Propylea japonica

RNA interference (RNAi)-mediated control of the notorious pest Henosepilachna vigintioctopunctata is an emerging environment friendly research area. However, the characterization of key target genes in H. vigintioctopunctata is crucial for this. Additionally, assessing the risk of RNAi to nontarget organisms (NTOs) is necessary for environmental safety. In this study, the potential of RNAi technology in controlling H. vigintioctopunctata infestation has been investigated by the oral delivery of double-stranded RNA (dsRNA). The results revealed that the silencing of six genes, including HvABCH1, HvHel25E, HvProsbeta5, HvProsalpha6, HvProsbeta6, and HvSrp54k, was highly lethal to H. vigintioctopunctata. The LC50 values of the dsRNAs used to silence these six genes were found to be less than 13 ng/?L. Moreover, the use of the bacterially expressed dsRNAs caused high mortality in the lab and field populations of H. vigintioctopunctata. Further, administration of HvHel25E and HvSrp54k dsRNAs in the predatory lady beetle Propylea japonica confirmed no transcriptional or organismal levels effects. This risk-assessment result ensured no off-target RNAi effects on the NTOs. Overall, the findings of the study suggested that HvABCH1, HvHel25E, HvProsbeta5, HvProsalpha6, HvProsbeta6, and HvSrp54k can be novel promising molecular targets with high specificity for H. vigintioctopunctata management with negligible effects on the NTOs.

RNA interference (RNAi)-mediated control of the notorious pest Henosepilachna vigintioctopunctata is an emerging environment friendly research area. However, the characterization of key target genes in H. vigintioctopunctata is crucial for this. Additionally, assessing the risk of RNAi to nontarget organisms (NTOs) is necessary for environmental safety. In this study, the potential of RNAi technology in controlling H. vigintioctopunctata infestation has been investigated by the oral delivery of double-stranded RNA (dsRNA). The results revealed that the silencing of six genes, including HvABCH1, HvHel25E, HvProsbeta5, HvProsalpha6, HvProsbeta6, and HvSrp54k, was highly lethal to H. vigintioctopunctata. The LC50 values of the dsRNAs used to silence these six genes were found to be less than 13 ng/?L. Moreover, the use of the bacterially expressed dsRNAs caused high mortality in the lab and field populations of H. vigintioctopunctata. Further, administration of HvHel25E and HvSrp54k dsRNAs in the predatory lady beetle Propylea japonica confirmed no transcriptional or organismal levels effects. This risk-assessment result ensured no off-target RNAi effects on the NTOs. Overall, the findings of the study suggested that HvABCH1, HvHel25E, HvProsbeta5, HvProsalpha6, HvProsbeta6, and HvSrp54k can be novel promising molecular targets with high specificity for H. vigintioctopunctata management with negligible effects on the NTOs.