syn-tasiRnas targeting the coat protein of potato virus Y confer antiviral resistance in Nicotiana benthamiana.

Trans-acting small interfering RNAs (tasiRNAs) are 21-nt phased (phased siRNAs) resulting from successive DCL-catalyzed processing from the end of a double-stranded RNA substrate originating from the RDR of an AGO-catalyzed cleaved RNA at a micro RNA target site. Plant tasiRNAs have been synthesized to produce synthetic tasiRNAs (syn-tasiRNAs) targeting viral RNAs that confer viral resistance. In this study, we engineered syn-tasiRNAs to target potato virus Y (PVY) infection by replacing five native siRNAs of TAS1c with 210-bp fragments from the coat protein (CP) region of the PVY genome. The results showed that the transient expression of syn-tasiR-CPpvy2 in Nicotiana benthamiana (N. benthamiana) plants conferred antiviral resistance, supported by the absence of PVY infection symptoms and viral accumulation. This indicated that syn-tasiR-CPpvy2 successfully targeted and silenced the PVY CP gene, effectively inhibiting viral infection. syn-tasiR-CPpvy1 displayed attenuated symptoms and decreased viral accumulation in these plants However, severe symptoms of PVY infection and a similar amount of viral accumulation as the control were observed in plants expressing syn-tasiR-CPpvy3. syn-tasiR-CPpvy/pvx, which targets both PVY and potato virus X (PVX), was engineered using a single precursor. After the transient expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 in N. benthamiana, the plants were resistant to both PVY and PVX. These results suggested that engineered syn-tasiRNAs could not only specifically induce antiviral resistance against one target virus but could also be designed for multi-targeted silencing of different viruses, thereby preventing complex virus infection in plants.

Transcriptome analysis and functional verification reveal the roles of copper in resistance to potato virus Y infection in tobacco.

Potato virus Y (PVY) is one of the most important pathogens in the genus Potyvirus that seriously harms agricultural production. Copper (Cu), as a micronutrient, is closely related to plant immune response. In this study, we found that foliar application of Cu could inhibit PVY infection to some extent, especially at 7 days post inoculation (dpi). To explore the effect of Cu on PVY infection, transcriptome sequencing analysis was performed on PVY-infected tobacco with or without Cu application. Several key pathways regulated by Cu were identified, including plant-pathogen interaction, inorganic ion transport and metabolism, and photosynthesis. Moreover, the results of virus-induced gene silencing (VIGS) assays revealed that NbMLP423, NbPIP2, NbFd and NbEXPA played positive roles in resistance to PVY infection in Nicotiana benthamiana. In addition, transgenic tobacco plants overexpressing NtEXPA11 showed increased resistance to PVY infection. These results contribute to clarify the role and regulatory mechanism of Cu against PVY infection, and provide candidate genes for disease resistance breeding.

Functional Characterization of Replication-Associated Proteins Encoded by Alphasatellites Identified in Yunnan Province, China.

Alphasatellites, which encode only a replication-associated protein (alpha-Rep), are frequently found to be non-essential satellite components associated with begomovirus/betasatellite complexes, and their presence can modulate disease symptoms and/or viral DNA accumulation during infection. Our previous study has shown that there are three types of alphasatellites associated with begomovirus/betasatellite complexes in Yunnan province in China and they encode three corresponding types of alpha-Rep proteins. However, the biological functions of alpha-Reps remain poorly understood. In this study, we investigated the biological functions of alpha-Reps in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) using 16c and 16-TGS transgenic Nicotiana benthamiana plants. Results showed that all the three types of alpha-Rep proteins were capable of suppressing the PTGS and reversing the TGS. Among them, the alpha-Rep of Y10DNA1 has the strongest PTGS and TGS suppressor activities. We also found that the alpha-Rep proteins were able to increase the accumulation of their helper virus during coinfection. These results suggest that the alpha-Reps may have a role in overcoming host defense, which provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus/betasatellite complexes.

Cauliflower mosaic virus P6 Dysfunctions Histone Deacetylase HD2C to Promote Virus Infection.

Histone deacetylases (HDACs) are vital epigenetic modifiers not only in regulating plant development but also in abiotic- and biotic-stress responses. Though to date, the functions of HD2C-an HD2-type HDAC-In plant development and abiotic stress have been intensively explored, its function in biotic stress remains unknown. In this study, we have identified HD2C as an interaction partner of the Cauliflower mosaic virus (CaMV) P6 protein. It functions as a positive regulator in defending against CaMV infection. The hd2c mutants show enhanced susceptibility to CaMV infection. In support, the accumulation of viral DNA, viral transcripts, and the deposition of histone acetylation on the viral minichromosomes are increased in hd2c mutants. P6 interferes with the interaction between HD2C and HDA6, and P6 overexpression lines have similar phenotypes with hd2c mutants. In further investigations, P6 overexpression lines, together with CaMV infection plants, are more sensitive to ABA and NaCl with a concomitant increasing expression of ABA/NaCl-regulated genes. Moreover, the global levels of histone acetylation are increased in P6 overexpression lines and CaMV infection plants. Collectively, our results suggest that P6 dysfunctions histone deacetylase HD2C by physical interaction to promote CaMV infection.

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Characterization of the Roles of SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 in Sw-5b-Mediated Resistance to Tomato Spotted Wilt Virus.

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways.

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication.

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH-dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC-mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2-Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC-mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.

Antiviral Activity of Ribosome-Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.

The hawthorn CpLRR-RLK1 gene targeted by ACLSV-derived vsiRNA positively regulate resistance to bacteria disease.

Virus-derived small interfering RNAs (vsiRNAs) can target not only viruses but also plant genes. Apple chlorotic leaf spot virus (ACLSV) is an RNA virus that infects Rosaceae plants extensively, including apple, pear and hawthorn. Here, we report an ACLSV-derived vsiRNA [vsiR1360(-)] that targets and down-regulates the leucine-rich repeat receptor-like kinase 1 (LRR-RLK1) gene of hawthorn (Crataegus pinnatifida). The targeting and cleavage of the CpLRR-RLK1 gene by vsiR1360(-) were validated by RNA ligase-mediated 5' rapid amplification of cDNA ends and tobacco transient transformation assays. And the CpLRR-RLK1 protein fused to green fluorescent protein localized to the cell membrane. Conserved domain and phylogenetic tree analyses showed that CpLRR-RLK1 is closely related to the proteins of the LRRII-RLK subfamily. The biological function of CpLRR-RLK1 was explored by heterologous overexpression of CpLRR-RLK1 gene in Arabidopsis. The results of inoculation of Pst DC3000 in Arabidopsis leaves showed that the symptoms of CpLRR-RLK1 overexpression plants infected with Pst DC3000 were significantly reduced compared with the wild type. In addition, the detection of reactive oxygen species and callose deposition and the expression analysis of defense-related genes showed that the CpLRR-RLK1 gene can indeed enhance the resistance of Arabidopsis to bacteria disease.

The C4 protein encoded by tomato leaf curl Yunnan virus reverses transcriptional gene silencing by interacting with NbDRM2 and impairing its DNA-binding ability.

In plants, cytosine DNA methylation is an efficient defense mechanism against geminiviruses, since methylation of the viral genome results in transcriptional gene silencing (TGS). As a counter-defense mechanism, geminiviruses encode viral proteins to suppress viral DNA methylation and TGS. However, the molecular mechanisms by which viral proteins contribute to TGS suppression remain incompletely understood. In this study, we found that the C4 protein encoded by tomato leaf curl Yunnan virus (TLCYnV) suppresses methylation of the viral genome through interacting with and impairing the DNA-binding ability of NbDRM2, a pivotal DNA methyltransferase in the methyl cycle. We show that NbDRM2 catalyzes the addition of methyl groups on specific cytosine sites of the viral genome, hence playing an important role in anti-viral defense. Underscoring the relevance of the C4-mediated suppression of NbDRM2 activity, plants infected by TLCYnV producing C4(S43A), a point mutant version of C4 unable to interact with NbDRM2, display milder symptoms and lower virus accumulation, concomitant with enhanced viral DNA methylation, than plants infected by wild-type TLCYnV. Expression of TLCYnV C4, but not of the NbDRM2-interaction compromised C4(S43A) mutant, in 16c-TGS Nicotiana benthamiana plants results in the recovery of GFP, a proxy for suppression of TGS. This study provides new insights into the molecular mechanisms by which geminiviruses suppress TGS, and uncovers a new viral strategy based on the inactivation of the methyltransferase NbDRM2.

Stability of Begomoviral pathogenicity determinant ßC1 is modulated by mutually antagonistic SUMOylation and SIM interactions.

BACKGROUND: To successfully invade new hosts, plant viruses must break host resistance and be competent to move within and between plant cells. As a means, viral proteins known as pathogenicity determinants have evolved to coordinate a network of protein interactions. The ßC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant for this disease-causing family of plant viruses. Post-translational modifications (PTMs) such as ubiquitination and phosphorylation of the ßC1 protein have been shown to occur in diverse viruses. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood. RESULTS: Here we identified the significance of a novel layer of PTMs in the ßC1 protein of Synedrella yellow vein clearing virus (SyYVCV), a newly identified member of the Begomovirus genus of Geminiviruses. This protein has conserved SUMOylation and SUMO-interacting motifs (SIMs), and we observed SUMOylation of SyYVCV ßC1 in host plants as a defensive strategy against ubiquitin-mediated degradation. Counteracting this, SIMs encoded in ßC1 mediate the degradation of ßC1; however, both these PTMs are essential for the function of ßC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. SUMOylation in different motifs of ßC1 led to functionally distinct outcomes, regulating the stability and function of the ßC1 protein, as well as increased global SUMOylation of host proteins. CONCLUSION: Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and, as a counter-defence, ßC1 undergoes SUMOylation to escape from its degradation.

Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato.

KEY MESSAGE: Expression of artificial microRNA targeting ATP binding domain of AC1 in transgenic tomato confers resistance to Tomato leaf curl disease without impacting the yield of tomato. Tomato curl leaf disease caused by Tomato leaf curl virus (ToLCV) is a key constraint to tomato cultivation worldwide. Engineering transgenic plants expressing artificial microRNAs (amiRNAs) against the AC1 gene of Tomato leaf curl New Delhi virus (ToLCNDV), which is important for virus replication and pathogenicity, would consequently confer virus resistance and reduce crop loss in the economically important crops. This study relates to an amiRNA developed on the sequence of Arabidopsis miRNA319a, targeting the ATP/GTP binding domain of AC1 gene of ToLCNDV. The AC1-amiR was found to regulate the abundance of AC1, providing an excellent strategy in providing defense against ToLCNDV. Transgenic lines over-expressing AC1-amiR, when challenged with ToLCNDV, showed reduced disease symptoms and high percentage resistance ranging between ∼ 40 and 80%. The yield of transgenic plants was significantly higher upon ToLCNDV infection as compared to the non-transgenic plants. Although the natural resistance resources against ToLCNDV are not available, this work streamlines a novel amiRNA-based mechanism that may have the potential to develop viral resistance strategies in tomato, apart from its normal symptom development properties as it is targeting the conserved region against which higher accumulation of small interfering RNAs (siRNA) occurred in a naturally tolerant tomato cultivar.

Localization of Tobacco Yellow Dwarf Virus Replication Using the In Plant Activation (INPACT) Expression Platform.

Geminiviruses and their diseases are a considerable economic threat to a vast number of crops worldwide. Investigating how and where these viruses replicate and accumulate in their hosts may lead to novel molecular resistance strategies. In this study, we used the Rep-inducible In Plant Activation (INPACT) expression platform, based on the genome of tobacco yellow dwarf virus (TYDV), to determine where this model mastrevirus replicates in its host tobacco. By developing an infectious clone of TYDV and optimizing its delivery by agroinfiltration, we first established an efficient artificial infection process. When delivered into transgenic tobacco plants containing a TYDV-based INPACT cassette encoding the ß-glucuronidase (GUS) reporter, we showed the virus activates GUS expression. Histology revealed that reporter gene expression was limited to phloem-associated cell types suggesting TYDV replication has a restricted tissue tropism.

Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus Infection in Arabidopsis and Soybean.

The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. We have previously shown that replacing seven amino acids at the cleavage site of PBS1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell-death response too slow to contain the virus. We theorized this could result from a positional mismatch within the cell between PBS1TuMV, RPS5, and the NIa protease. To test this, we relocalized PBS1TuMV and RPS5 to cellular sites of NIa accumulation. These experiments revealed that relocation of RPS5 away from the plasma membrane compromised RPS5-dependent cell death in Nicotiana benthamiana, even though PBS1 was efficiently cleaved. As an alternative approach, we tested whether overexpression of plasma membrane-localized PBS1TuMV could enhance RPS5 activation by TuMV. Significantly, overexpressing the PBS1TuMV decoy protein conferred complete resistance to TuMV when delivered by either agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens. Lastly, we have now extended this PBS1 decoy approach to soybean by modifying a soybean PBS1 ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that we can use endogenous PBS1 proteins in crop plants to engineer economically relevant disease resistant traits.

Foliar exposure of Fe3O4 nanoparticles on Nicotiana benthamiana: Evidence for nanoparticles uptake, plant growth promoter and defense response elicitor against plant virus.

Nanoparticles are recently employed as a new strategy to directly kill pathogens (e.g., bacteria and fungus) and acted as nanofertilizers. However, the influences of this foliar deposition of nanoparticles on plant physiology particularly plant immunity are poorly understood. The uptake and physiological effects of Fe3O4 nanoparticles (Fe3O4NPs), and plant resistance response against Tobacco mosaic virus (TMV) after foliar spraying were studied. Specifically, Fe3O4NPs entered leaf cells and were transported and accumulated throughout the whole Nicotiana benthamiana plant, and increased plant dry and fresh weights, activated plant antioxidants, and upregulated SA synthesis and the expression of SA-responsive PR genes (i.e., PR1 and PR2), thereby enhancing plant resistance against TMV. Conversely, the viral infection was not inhibited in the NahG transgenic plants treated by Fe3O4NPs, suggesting the involvement of salicylic acid (SA) induced by Fe3O4NPs in the production of plant resistance. Moreover, no inhibition was observed of the infection after inoculating with the pretreated TMV mixtures. Thus, the deposition of Fe3O4NPs induced the accumulation of endogenous SA, which was correlated with the plant resistance against TMV infection. Such information is vital for valuing the risk of Fe3O4NPs products and broadens the researching and applying nanoparticles in the fight against plant diseases meantime.

LreEF1A4, a Translation Elongation Factor from Lilium regale, Is Pivotal for Cucumber Mosaic Virus and Tobacco Rattle Virus Infections and Tolerance to Salt and Drought.

Eukaryotic translation elongation factors are implicated in protein synthesis across different living organisms, but their biological functions in the pathogenesis of cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) infections are poorly understood. Here, we isolated and characterized a cDNA clone, LreEF1A4, encoding the alpha subunit of elongation factor 1, from a CMV-elicited suppression subtractive hybridization library of Lilium regale. The infection tests using CMV remarkably increased transcript abundance of LreEF1A4; however, it also led to inconsistent expression profiles of three other LreEF1A homologs (LreEF1A1-3). Protein modelling analysis revealed that the amino acid substitutions among four LreEF1As may not affect their enzymatic functions. LreEF1A4 was ectopically overexpressed in petunia (Petunia hybrida), and transgenic plants exhibited delayed leaf and flower senescence, concomitant with increased transcription of photosynthesis-related genes and reduced expression of senescence-associated genes, respectively. A compromised resistance to CMV and TRV infections was found in transgenic petunia plants overexpressing LreEF1A4, whereas its overexpression resulted in an enhanced tolerance to salt and drought stresses. Taken together, our data demonstrate that LreEF1A4 functions as a positive regulator in viral multiplication and plant adaption to high salinity and dehydration.

Conformational behavior of coat protein in plants and association with coat protein-mediated resistance against TMV.

Tobacco mosaic virus (TMV) coat protein (CP) self assembles in viral RNA deprived transgenic plants to form aggregates based on the physical conditions of the environment. Transgenic plants in which these aggregates are developed show resistance toward infection by TMV referred to as CP-MR. This phenomenon has been extensively used to protect transgenic plants against viral diseases. The mutants T42W and E50Q CP confer enhanced CP-MR as compared to the WT CP. The aggregates, when examined, show the presence of helical discs in the case of WT CP; on the other hand, mutants show the presence of highly stable non-helical long rods. These aggregates interfere with the accumulation of MP as well as with the disassembly of TMV in plant cells. Here, we explored an atomic level insight to the process of CP-MR through MD simulations. The subunit-subunit interactions were assessed with the help of MM-PBSA calculations. Moreover, classification of secondary structure elements of the protein also provided unambiguous information about the conformational changes occurring in the two chains, which indicated toward increased flexibility of the mutant protein and seconded the other results of simulations. Our finding indicates the essential structural changes caused by the mutation in CP subunits, which are critically responsible for CP-MR and provides an in silico insight into the effects of these transitions over CP-MR. These results could further be utilized to design TMV-CP-based small peptides that would be able to provide appropriate protection against TMV infection.

Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex.

Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield.The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex. It was found that transgenic plants over expressing Hydroperoxidelyase (HPL) genes exhibited higher enzyme activity than wild type. In addition the genome sequence information was used for the purpose of gene isolation. Even for the enhanced expression of Calreticulin (CRT), AOS and HPL in G. hirsutum, it still showed susceptibility against CLCuV suggesting alternative genes and pathways involved for the expression of resistance.

Artificially induced phased siRNAs promote virus resistance in transgenic plants.

We previously developed transgenic tobacco plants that were resistant to two geminiviruses. We generated resistance using RNAi constructs that produced trans-acting siRNA (tasiRNA) like secondary siRNAs known as phased siRNA (phasiRNA) that targeted several regions of Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Tomato Leaf Curl Gujarat Virus (ToLCGV) transcripts encoding the RNA silencing suppressor proteins AC2 and AC4. Here, we performed degradome analysis to determine the precise cleavage sites of RNA-RNA interaction between phasiRNA and viral transcripts. We then applied our RNAi technology in tomato, which is the natural host for ToLCNDV and ToLCGV. The relative ease of developing and using phasiRNA constructs represents a significant technical advance in imparting virus resistance in crops and/or important model systems.