RNAi-based silencing of proteasome 20S subunit alpha 2 affected the survival and development of Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is a notorious pest of solanaceous plants in Asia, which is mainly managed by chemical pesticides. RNA interference (RNAi) technique is considered to be a promising and effective alternative for pest control. In this study, we selected the proteasome 20S subunit alpha 2 (Prosα2) gene, a cellular protein involved in many proteins regulatory processes, to explore the RNAi efficiency in H. vigintioctopunctata. The obtained results confirmed the significant lethal effects of HvProsα2 silencing on the H. vigintioctopunctata 1st instar larvae at concentrations of 100, 50, and 5 ng/µL. Ingestion of the bacterially expressed dsHvProsα2 caused high mortality in both larvae and adults. Moreover, silencing of HvProsα2 resulted in feeding disorders, growth delay, and abnormal intestinal development of the larvae. Overall, HvProsα2 acts as an important regulator for the growth and development of H. vigintioctopunctata, and can serve as a candidate target gene for the RNAi-based control of H. vigintioctopunctata.

Unveiling Nilaparvata lugens Stål Genes Defining Compatible and Incompatible Interactions with Rice through Transcriptome Analysis and Gene Silencing.

The brown planthopper (Nilaparvata lugens Stål, BPH) is a major pest of rice (Oryza sativa L.), causing severe crop loss. Multiple biotypes and emerging populations of BPH pose a bigger challenge for the infestations control. Although several studies have been conducted to understand the molecular mechanisms of rice-BPH interactions, there are few studies dedicated to the Indian sub-continent BPH biotype (biotype 4). Here, we analyzed the transcriptomic, physiological, and gene-silencing responses of the BPH biotype 4 during the compatible (fed on susceptible Taichung Native 1, TN1 rice) and incompatible (fed on resistant PTB33 rice) rice-BPH interactions. In the incompatible interaction, a significant reduction in the honeydew production and negative weight gain were observed in the BPH. Similarly, the trehalose and glucose contents were found to be significantly high and low, respectively, during the incompatible rice-BPH interaction. The comparative BPH transcriptome analysis identified 1875 differentially expressive genes (DEGs) between the compatible and incompatible interactions from which many were annotated to be involved in vital BPH physiological processes, including cuticle development, sugar metabolism, detoxification, molting, and xenobiotics metabolism. The RNA interference-mediated independent silencing of three selected genes, including NlCP1, NlCYP320a1, and NlTret1, revealed that these genes are important for BPH physiology and survival. Moreover, the results of this study provide valuable insights into the rice-BPH interactions involving the BPH biotype 4.

Colonization of Serendipita indica promotes resistance against Spodoptera exigua in onion (Allium cepa L.).

Plant-endophyte symbiosis influences plant defense and growth. Serendipita indica is a root endophyte that promotes growth and induces tolerance against biotic and abiotic stress in plants. In this study, we examined the effect of S. indica colonization on herbivore (Spodoptera exigua) resistance of onion (Allium cepa L.). We found that colonization of S. indica in the roots of onion significantly reduced the feeding damage of leaves by S. exigua larvae, and also resulted in a reduction in weight gain of the larvae when fed on S. indica plants. This enhanced resistance is a result of modulation of antioxidant and defense enzymes/genes in the host by S. indica mutualism. Specifically, the activities of enzymes such as Superoxide dismutase, peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, and H2O2 content were significantly higher in the early stages of S. exigua feeding in the S. indica colonized plants compared to the non-colonized counterparts. Similarly, defense genes also showed modulation in response to this tripartite interaction of onion -S. indica mutualism and S. exigua herbivory. The hierarchical cluster analysis and principal component analysis indicated a clear difference in the onion biochemical responses, which is due to the S. indica symbiosis. Our investigation demonstrates that onion-S. indica symbiosis significantly decreases chewing injury by efficiently modulating antioxidant and defense enzyme activities and gene expression in response to S. exigua herbivory. Therefore, S. indica can be used as a potential biocontrol agent for sustainable management of this important pest of Alliums.

RNAi assays in the striped flea beetle (Phyllotreta striolata) suggest Psγ-COPI and PsArf1COPI as potential molecular targets for pest control.

Phyllotreta striolata (Fabricius), commonly known as the striped flea beetle (SFB), is a notorious insect pest that attacks Brassicaceae plants worldwide, leading to tremendous economic losses. RNA interference (RNAi) has been proposed as a promising strategy for sustainable and eco-friendly pest control. In this study, a total of nine housekeeping genes including PsVATPA, PsHSP90, PsEF1A, PsRPL6, PsRPS24, PsActin, PsTUBA, PsRPS18, and PsRPL4 were evaluated under four different conditions (organization, population, sex, and RNAi). PsEF1A and PsVATPA were identified as the best reference genes for RNAi bioassay. Furthermore, a total of 24 target genes were selected to investigate their RNAi effects in SFB adults with double-stranded RNAs (dsRNAs), five of them showed significant mortality (28.00% to 70.00%), namely Psα-COPI, Psß-COPI, PsRPS18, Psγ-COPI, and PsArf1COPI. We found that gene transcript levels of the two most lethal genes, Psγ-COPI and PsArf1COPI, were significantly decreased after treated with the target dsRNAs either by feeding or injection method. The findings from this study demonstrated that the introduction of dsRNAs via oral feedings or injection induces the RNAi-mediated silencing of target genes and can lead to insect mortality. Overall, the identified target genes can be explored in developing RNAi-based insecticides for SFB control.

Genome-wide identification and molecular characterization of CRK gene family in cucumber (Cucumis sativus L.) under cold stress and sclerotium rolfsii infection.

BACKGROUND: The plant cysteine-rich receptor-like kinases (CRKs) are a large family having multiple roles, including defense responses under both biotic and abiotic stress. However, the CRK family in cucumbers (Cucumis sativus L.) has been explored to a limited extent. In this study, a genome-wide characterization of the CRK family has been performed to investigate the structural and functional attributes of the cucumber CRKs under cold and fungal pathogen stress. RESULTS: A total of 15 C. sativus CRKs (CsCRKs) have been characterized in the cucumber genome. Chromosome mapping of the CsCRKs revealed that 15 genes are distributed in cucumber chromosomes. Additionally, the gene duplication analysis of the CsCRKs yielded information on their divergence and expansion in cucumbers. Phylogenetic analysis divided the CsCRKs into two clades along with other plant CRKs. Functional predictions of the CsCRKs suggested their role in signaling and defense response in cucumbers. The expression analysis of the CsCRKs by using transcriptome data and via qRT-PCR indicated their involvement in both biotic and abiotic stress responses. Under the cucumber neck rot pathogen, Sclerotium rolfsii infection, multiple CsCRKs exhibited induced expressions at early, late, and both stages. Finally, the protein interaction network prediction results identified some key possible interacting partners of the CsCRKs in regulating cucumber physiological processes. CONCLUSIONS: The results of this study identified and characterized the CRK gene family in cucumbers. Functional predictions and validation via expression analysis confirmed the involvement of the CsCRKs in cucumber defense response, especially against S. rolfsii. Moreover, current findings provide better insights into the cucumber CRKs and their involvement in defense responses.

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 µM concentration) and BR (at 0 and 5 µM 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.

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/µL. 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.

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.

Tyrosine hydroxylase 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. © 2022 Society of Chemical Industry.

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 (FTZF1) 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 20E-related 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, HvFTZ-F1 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.

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. © 2021 Society of Chemical Industry.

Auxin and Cytokinin Interplay during Leaf Morphogenesis and Phyllotaxy.

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.

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.

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.

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.

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.

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.

The Roles of E93 and Kr-h1 in 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.

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.

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.