Small Heat Shock Protein (sHsp22.98) from Trialeurodes vaporariorum Plays Important Role in Apple Scar Skin Viroid Transmission.

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly's proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.

Two light responsive WRKY genes exhibit positive and negative correlation with picroside content in Picrorhiza kurrooa Royle ex Benth, an endangered medicinal herb.

Picrorhiza kurrooa is an endangered herb known to produce the medicinally important picrosides through isoprenoid pathway. The present work showed the functionality of WRKY motifs (TGAC cis-acting elements) present in the promoters of regulatory genes 3-hydroxy-3-methylglutaryl coenzyme A reductase (Pkhmgr) and 1-deoxy-d-xylulose-5-phosphate synthase (Pkdxs) of the picrosides biosynthetic pathway by electrophoretic mobility shift assay. Also, the two WRKY genes, PkdWRKY and PksWRKY, were characterized and found to contain double and single characteristic WRKY domains, respectively along with a zinc-finger motif in each domain. Expression analysis revealed that PkdWRKY and PksWRKY exhibited a positive and negative correlation, respectively, with picrosides content under the environment of light and in different tissues. Functional evaluation in yeast showed DNA binding ability of both PksWRKY and PkdWRKY; however, only PkdWRKY exhibited transcriptional activation ability. Transient overexpression of PkdWRKY and PksWRKY in tobacco modulated the expression of selected native genes of tobacco involved in MVA and MEP pathway suggesting functionality of PkdWRKY and PksWRKY in planta. Collectively, data suggested that PkdWRKY and PksWRKY might be positive and negative regulators, respectively in the picrosides biosynthetic pathway.

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants.

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

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.

AC4 protein of tomato leaf curl Palampur virus is an RNA silencing suppressor and a pathogenicity determinant.

Plants deploy RNA silencing as a natural defence against invading viruses involving sequence-specific degradation of the viral RNAs. As a counter-defence strategy, viruses encode suppressor proteins that simultaneously target different steps of the silencing machinery. Tomato leaf curl Palampur virus (ToLCPalV) is a bipartite begomovirus in Geminiviridae family. It is responsible for significant reduction in the crop yield and quality. DNA-A of the virus encodes for six proteins whereas DNA-B codes for two proteins. In this study, all viral genes were screened for their role in suppression of green fluorescent protein (GFP) silencing in Nicotiana tabacum cv. Xanthi, employing agrobacterium based co-infiltration assay. The assay identified AC4 as a potential suppressor of RNA silencing. In addition, AC4 expression also suppressed virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in N. benthamiana. Potato virus X (PVX) mediated transient expression of the AC4 in N. benthamiana showed enhanced symptoms that include downward leaf curling, leaf puckering and tissue necrosis. Further, N. benthamiana lines stably expressing AC4 showed severe developmental abnormalities. Mutational analysis suggested that glycine at 2nd position is essential for AC4 pathogenicity. Collectively, these findings demonstrate the role of ToLCPalV AC4 in viral pathogenesis, disease establishment and suppression of gene silencing.

AV2 protein of tomato leaf curl Palampur virus promotes systemic necrosis in Nicotiana benthamiana and interacts with host Catalase2.

Tomato leaf curl Palampur virus (ToLCPalV) is a whitefly-transmitted, bipartite begomovirus. Here, we demonstrated that ectopic expression of AV2 from a Potato virus X (PVX)-based vector accelerated systemic necrosis and reactive oxygen species (ROS) accumulation in Nicotiana benthamiana. Furthermore, 10 amino acids from N-terminal region of AV2 were found to be associated with the systemic necrosis symptom/phenotype. Mutational studies of ToLCPalV infectious clones lacking the AV2 revealed that AV2 is essential for the systemic movement of DNA-A, symptom severity and viral DNA accumulation. In a yeast two-hybrid assay, Catalase2 (Cat2) was found to associate with AV2 protein. Further, silencing of Cat2 resulted in appearance of necrotic lesions on N. benthamiana and these plants were highly susceptible to ToLCPalV infection in comparison to control plants. Infection ToLCPalV on Solanum lycopersicum resulted in downregulation of Cat2 transcripts, followed by accumulation of ROS and stress marker transcripts. The AV2 protein also suppressed virus-induced gene silencing (VIGS) of the Phytoene desaturase (PDS) gene. Our results show that AV2 is essential for the pathogenicity, systemic movement and suppression of gene silencing in the host. Altogether, our findings suggest that interactions between AV2 and Cat2 might play a crucial role in the establishment of ToLCPalV infection.

Molecular characterization of a new begomovirus infecting Mirabilis jalapa in northern India.

Begomoviruses are whitefly-transmitted single-stranded DNA viruses that are responsible for considerable economic losses. A begomovirus, alphasatellite and betasatellite were characterized in a Mirabilis jalapa plant exhibiting severe leaf curling and mottling symptoms. The complete viral genome shared highest sequence identity of 87% with pedilanthus leaf curl virus (AM712436), reported from Pakistan. Additionally, the viral genome was 84% identical to that of chilli leaf curl India virus (KX951415) and 83% identical to that of tobacco curly shoot virus (GU1999584), which were previously reported to infect M. jalapa in India and China, respectively. Based on the ICTV criterion for begomovirus species demarcation (≥91% sequence identity for the complete genome), the virus represents a new species, for which we propose the name Mirabilis leaf curl virus. The alphasatellite and betasatellite sequences were similar to the corresponding sequences of ageratum yellow vein India alphasatellite (KU852743; 99% identity) and tomato leaf curl Patna betasatellite (HQ180394; 86% identity) sequences, respectively. This report describes a new begomovirus-satellite disease complex in M. jalapa.

Identification of host cellular targets of AC4 and AV2 proteins of tomato leaf curl palampur virus and their sub-cellular localization studies.

Tomato leaf curl palampur virus (ToLCPalV) is a bipartite begomovirus with genome organization typical of old world begomoviruses. It infects commercially important crops and weeds in the Asian subcontinent. Apart from other proteins, the DNA-A of the virus encodes AV2 and AC4 proteins of approximately 13.73 and 6.7 kDa, respectively. In case of other begomoviruses, previous studies have shown the role of AV2 and AC4 proteins in virus movement, pathogenesis and suppression of gene silencing. However, the ToLCPalV proteins are significantly variable in comparison to closest relative and hence there is a need to work out their functions. In this study, we identified 9 cellular proteins of tomato that interact with AV2 and AC4 proteins, through yeast two hybrid screening. Upon sequence analysis, these interactors were identified as cysteine protease, katanin p60 ATPase-containing subunit A-like, guanine deaminase, NADH dehydrogenase (ubiquinone) iron-sulfur protein, glyceraldehyde-3-phosphate dehydrogenase B, 60S acidic ribosomal P0 protein, acyl co-A dehydrogenase IBR3, oxygen-evolving enhancer protein 1 and peroxisomal membrane protein 11D. These proteins play a vital role in protein degradation, plant defense response, microtubule severing, photosynthesis and protein synthesis. The two viral proteins, however, did not interact with each other in yeast. AV2 when fused with GFP under the control of cauliflower mosaic virus 35S promoter was localized in nucleus and cytoplasm. On the other hand, AC4-GFP fusion was localized only in cytoplasm. The outcome of present study will help to elucidate the mechanism of viral pathogenesis. Further functional characterization of identified host proteins will provide an insight into their involvement in disease development.

Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase.

Plant viral movement proteins facilitate virion movement mainly through interaction with a number of factors from the host. We report the association of a cell wall localized ascorbate oxidase (CsAO4) from Cucumis sativus with the movement protein (MP) of Cucumber mosaic virus (CMV). This was identified first in a yeast two-hybrid screen and validated by in vivo pull down and bimolecular fluorescence complementation (BiFC) assays. The BiFC assay showed localization of the bimolecular complexes of these proteins around the cell wall periphery as punctate spots. The expression of CsAO4 was induced during the initial infection period (up to 72 h) in CMV infected Nicotiana benthamiana plants. To functionally validate its role in viral spread, we analyzed the virus accumulation in CsAO4 overexpressing Arabidopsis thaliana and transiently silenced N. benthamiana plants (through a Tobacco rattle virus vector). Overexpression had no evident effect on virus accumulation in upper non-inoculated leaves of transgenic lines in comparison to WT plants at 7 days post inoculation (dpi). However, knockdown resulted in reduced CMV accumulation in systemic (non-inoculated) leaves of NbΔAO-pTRV2 silenced plants as compared to TRV inoculated control plants at 5 dpi (up to 1.3 fold difference). In addition, functional validation supported the importance of AO in plant development. These findings suggest that AO and viral MP interaction helps in early viral movement; however, it had no major effect on viral accumulation after 7 dpi. This study suggests that initial induction of expression of AO on virus infection and its association with viral MP helps both towards targeting of the MP to the apoplast and disrupting formation of functional AO dimers for spread of virus to nearby cells, reducing the redox defense of the plant during initial stages of infection.

Molecular identification of Ageratum enation virus, betasatellite and alphasatellite molecules isolated from yellow vein diseased Amaranthus cruentus in India.

Natural occurrence of yellow vein disease on Amaranthus cruentus was observed at Lucknow, India in the year 2008. The causal virus was successfully transmitted through whiteflies (Bemisia tabaci) from diseased A. cruentus to healthy seedlings of A. cruentus and other test species which indicated begomovirus infection. The begomovirus DNA-A, betasatellite, and alphasatellite components associated with yellow vein disease were amplified by rolling circle amplification using Ø-29 DNA polymerase from diseased A. cruentus and characterized by their sequence analyses. The begomovirus DNA-A genome contained six ORFs: AV2 and AV1 in virion sense and AC3, AC2, AC1, and AC4 in complementary sense strand; and a non-translated intergenic region having the conserved geminiviral nonanucleotide sequence. The virus isolate showed 97-99% sequence identities and close phylogenetic relationships with various isolates of Ageratum enation virus (AgEV); therefore, the isolate under study was identified as AgEV. The beta- and alphasatellite molecules were also identified to be associated with the disease based on their high sequence identities and close phylogenetic relationships with the respective molecules reported worldwide. Co-infiltration of agro-infectious clones of AgEV DNA-A and its betasatellite DNA induced leaf curl and enation symptoms after 25-35 days on A. cruentus, Nicotiana benthamiana, and N. glutinosa plants. We report the association of AgEV, betasatellite and alphasatellite components with yellow vein disease of A. cruentus from India.

Biological and Molecular Characterization of Cucumber mosaic virus Subgroup II Isolate Causing Severe Mosaic in Cucumber.

Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricultural and ornamental crops. Earlier reports showed the prevalence of CMV subgroup I isolates in India. However, some recent reports point towards increasing incidence of subgroup II isolates in the country. The complete genome of a CMV isolate causing severe mosaic in cucumber was characterized and its phylogenetic analysis with other 21 CMV isolates reported worldwide clustered it with subgroup II strains. The genome comprised of RNA 1 (3,379 nucleotides), RNA 2 (3,038 nucleotides) and RNA 3 (2,206 nucleotides). The isolate showed highest homology with subgroup II isolates: 95.1-98.7, 87.7-98.0, and 85.4-97.1 % within RNA1, RNA2, and RNA3, respectively. RNA1 and RNA2 were closely related to the Japanese isolate while RNA3 clustered with an American isolate. Host range studies revealed that isolate showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The isolate induced leaf deformation and mild filiform type symptoms in tomato. To best of our knowledge this is the first report of complete genome of CMV subgroup II isolate from India.

Plant parasitic and vector nematodes associated with Asiatic and Oriental hybrid lilies.

A survey for distribution and abundance of plant parasitic nematodes in fields grown to Lilium in Himachal Pradesh, India at four study sites viz. Nagrota (at 810 m a.s.l.), Palampur (at 1270 m a.s.l.), Sunder Nagar (at 1400 m a.s.l.) and Chail (at 2250 m a.s.l.) was carried out. Moderate (101-500/200 ml soil) to high (501-1000/200 ml soil) populations of phytonematodes including the vectors for plant viruses (Aphelenchoides avenae, Criconemoides spp., Hoplolaimus spp., Longidorus spp., Paratylenchus spp., Pratylenchus spp., Rhabditis spp., Trichodorus spp., Tylenchoryhnchus spp., Tylenchulus spp. and Xiphinema diversicaudatum) were recorded. Mean population of nematodes was positively correlated with pH in all the study sites, negatively correlated with electrical conductivity (EC), percent organic matter (OM%), available potassium (K) and positively correlated with percent carbon (C%), available nitrogen (N) and available phosphorus (P) in all but one study site. The highest incidence of virus-vector nematodes viz. X. diversicaudatum, Longidorus spp. and Trichodorus spp. was recorded at Palampur. Only Strawberry latent ringspot nepovirus (SLRSV) was detected in Lilium cvs. Star Gazer Max and Galeili by Enzyme Linked Immunosorbent Assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) and in X. diversicaudatum associated with the cultivars by RT-PCR. Cucumis sativus used as bait plants showed SLRSV symptoms after 15 days of nematode inoculation and further SLRSV was again detected by ELISA and RT-PCR in C. sativus plants confirming the transmission of SLRSV by X. diversicaudatum in Lilium.