Protein-protein interactions and nuclear trafficking of coat protein and betaC1 protein associated with Bhendi yellow vein mosaic disease

Bhendi yellow vein mosaic disease (BYVMD) is caused by a complex consisting of a monopartite begomovirus BYVMV and a satellite DNA beta component. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction. Here we report the results of the transient expression of green fluorescent protein (GFP) fused with the betaC1 and coat protein (CP) coding regions, in the epidermal cells of Nicotiana benthamiana. GFPCP was found to be targeted into the nucleus whereas GFPbetaC1 was localized towards the periphery of the cell. The sub-cellular localization of the betaC1 protein has been compared with that of the CP in yeast cells using a genetic system for detection of protein nuclear import and export. Expression of betaC1 ORF in transgenic N. benthamiana under the control of the Cauliflower mosaic virus 35S promoter produced severe developmental abnormalities in the plant, like distorted stem, leaves and stunting of the plant. We also present the results on the interaction of CP and betaC1 proteins using yeast two hybrid analysis, suggesting a collaborative role in the inter- and intracellular dynamics of BYVMD.     

Contribution of the satellite encoded gene betaC1 to cotton leaf curl disease symptoms

Cotton leaf curl disease (CLCuD) is caused by one of seven begomoviruses in conjunction with a specific satellite; CLCuD DNA beta. Associated with some monopartite begomoviruses, DNA beta components encode a single gene (betaC1) which mediates satellite functions. We have investigated the contribution the satellite, specifically betaC1, makes to CLCuD symptoms in the absence of the helper begomovirus. Systemic expression of CLCuD-betaC1 from a Potato virus X (PVX) vector induces bona fide CLCuD disease symptoms in Nicotiana tabacum plants, including enations, swollen veins and vein darkening. These contrast with the mild symptoms of PVX in this host. Analysis of thin sections across enations induced by PVX expressing betaC1 shows the structure of the enation to be identical to those induced by CLCuD DNA beta in conjunction with a helper begomovirus. These results demonstrate that CLCuD betaC1 is the major determinant of symptoms for the CLCuD complex.     

Post-transcriptional gene silencing suppressor activity of two non-pathogenic alphasatellites associated with a begomovirus

Alphasatellites and betasatellites are begomovirus-associated single-stranded circular DNA molecules. Two distinct alphasatellites, Gossypium darwinii symptomless alphasatellite and Gossypium mustelinium symptomless alphasatellite, were previously isolated from Gossypium davidsonii and G.mustelinium. Here we show that the replication-associated proteins (Rep: a rolling-circle replication initiator protein) encoded by these alphasatellites interact with the Rep and C4 proteins encoded by their helper begomovirus, Cotton leaf curl Rajasthan virus (CLCuRaV), in a yeast two-hybrid assay. Both the alphasatellite-encoded Reps were found to have strong gene silencing suppressor activity, in contrast to the betasatellite-encoded βC1 and CLCuRaV-encoded C2, C4 and V2 proteins. The presence of alphasatellites maintained suppression of gene silencing in the youngest, actively growing tissue of CLCuRaV-betasatellite-infected plants. This is the first demonstration of a rolling-circle replication initiator protein with suppressor of gene silencing activity and provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus-betasatellite complexes.     

A modified viral satellite DNA-based gene silencing vector is effective in association with heterologous begomoviruses

We have previously reported effective gene silencing of a transgene and endogenous plant genes in tobacco and tomato plants using a modified viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV). In this study, we constructed a similar gene silencing vector (DNADeltaC12beta) based on the satellite DNAbeta associated with Tobacco curly shoot virus (TbCSV) by replacing its betaC1 gene with a multiple cloning site. Strong and stable silencing of cognate genes was achieved when this vector, carrying a fragment of the green fluorescent protein (GFP) transgene or a sulfur (Su) endogenous gene encoding one unit of the chloroplast enzyme magnesium chelatase required for chlorophyll II production, was co-agroinoculated with TbCSV used as a helper virus. GFP silenced transgenic Nicotiana benthamiana plants appear red under UV illumination due to loss of green fluorescence, while the Su silenced plants appear white as a result of failure to synthesize chlorophyll. Our results show that the efficiency of Su silencing is independent of the insert orientation in both N. benthamiana and N. glutinosa plants. Most significant however, is the observation that in association with heterologous begomoviruses, such as TYLCCNV or Malvastrum yellow vein virus, the DNADeltaC12beta vector could still effectively induce transgene and endogenous gene silencing in tobacco plants. These observations suggest that the modified viral satellite DNA vector can be applied as a reverse genetics tool for the study, analysis and discovery of gene function in more plants.     

Role of a geminivirus AV2 protein putative protein kinase C motif on subcellular localization and pathogenicity

Virus-derived genes or genome fragments are increasingly being used to generate transgenic plants with resistance to plant viruses. There is need to rapidly investigate these genes in plants using transient expression prior to using them as transgenes since they may be pathogenic to plants. In this study, we investigated the AV2 protein encoded by East African cassava mosaic Cameroon virus, a virus associated with a cassava disease epidemic in western Africa. For subcellular localization, AV2 was fused to the yellow fluorescent protein (YFP) and expressed in Nicotiana benthamiana. Confocal analyses showed that AV2-YFP localizes mainly in the cytoplasm. Because it overlaps with the coat protein gene and therefore could be used to generate transgenic plants for resistance to geminiviruses, we investigated its pathogenesis in N. benthamiana by using the Potato virus X (PVX) vector. The chimeric virus PVX-AV2 induced a mild mottling in infected plants and was shown to suppress virus-induced gene silencing (VIGS). Using point mutations, we show here that AV2 pathogenicity is dependent on a conserved putative protein kinase C (PKC) phosphorylation motif. Because of its pathogenicity and ability to suppress RNA silencing, AV2 transgenic plants will less likely provide a control to geminiviruses, indeed it may weaken the resistance of the plant. We therefore suggest the use of the AV2 putative PKC mutants to generate transgenic plants.     

Size reversion of a truncated DNAbeta associated with Tobacco curly shoot virus

Our previous results demonstrated that DNAbeta associated with Tobacco curly shoot virus (TbCSV) is not necessary for infection but intensifies symptoms in some hosts. To better understand the function of DNAbeta in virus infection, a betaC1 deleted infectious clone of the TbCSV DNAbeta was constructed. Agroinoculation showed that the truncated DNAbeta (DNADeltaC1beta) was trans-replicated by TbCSV in tobacco and Petunia hybrida plants. However, PCR and Southern blot analysis demonstrated that the truncated DNAbeta reverted to near wild type component size in some Nicotiana benthamiana, N. glutinosa, N. tabacum Samsun and P. hybrida plants co-inoculated with TbCSV and DNADeltaC1beta. Sequence analysis of four DNADeltaC1beta derivatives revealed that the wild type size DNAbeta molecules were recombinants between TbCSV DNAbeta and the pBinPLUS vector in which dimeric constructs were produced for inoculation. The significance of these findings is discussed with respect to the constraints imposed on begomovirus genome size.     

Cucumber necrosis virus p20 is a viral suppressor of RNA silencing

The p20 protein encoded by the tombusvirus, Cucumber necrosis virus has previously been shown to be involved in host pathogenicity and shares sequence similarity with the Tomato bushy stunt virus p19 suppressor of silencing. Using a virus-induced gene silencing (VIGS) assay, we show that p20 is a viral suppressor of RNA silencing (VSR) in infected plants. In addition, a CNV p20-knockout mutant showed a decline in viral RNA accumulation in infected plants, consistent with the role of p20 in suppression of RNA silencing. However, unexpectedly, all GFP transgenic plants co-infiltrated with p20 and GFP displayed RNA silencing using an Agrobacterium-mediated silencing assay. Detailed RNA analysis of GFP mRNA levels in p20 agro-infiltrated plants revealed that p20 did initially display suppressor activity but this was rapidly overcome by RNA silencing. p20 expression levels in agro-infiltrated plants were shown to be approximately 50-fold lower than that of the TBSV p19 silencing suppressor, consistent with the notion that p20 dosage levels are not sufficient to suppress RNA silencing in the Agrobacterium-mediated system. Our results suggest that a viral-based VIGS assay may be required for identifying VSRs encoded by some plant viruses. Based on bioinformatics studies the mechanism of suppression of silencing by p20 is predicted to be similar to that of the TBSV p19 suppressor.     

Cryptococcus neoformans differential gene expression detected in vitro and in vivo with green fluorescent protein

Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungus Cryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFalpha1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFalpha1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFalpha1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFalpha1 promoter is induced by nutrient deprivation and the MATalpha locus containing the MFalpha1 gene has been linked with virulence, yeast cells containing the pMFalpha1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFalpha1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFalpha1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.     

Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.     

Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters.

To test tissue specificity of zebrafish gene promoters in a heterologous fish species, two transgenic medaka lines under two zebrafish promoters were generated. Under the zebrafish skeletal muscle-specific mylz2 promoter, transgenic medaka expressed green fluorescent protein (GFP) exclusively in skeletal muscles, mimicking the endogenous medaka mylz2 mRNA expression and also identical to GFP expression in mylz2:gfp transgenic zebrafish. A madaka mylz2 promoter was also capable of directing skeletal muscle-specific GFP expression in transient transgenic zebrafish embryos. In the krt8:rfp transgenic medaka line with the zebrafish epithelial krt8 promoter, red fluorescent protein was specifically expressed in the skin epithelia as well as the epithelial lining cells of the anterior digestive tract, which was also identical to GFP expression in krt8:gfp transgenic zebrafish. Therefore, the two zebrafish promoters faithfully function in a heterologous fish species, and it is likely that the mechanisms of tissue-specific expression are largely conserved among fish species.     

Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs

To increase the efficiency of infections with Cowpea mosaic virus (CPMV)-based constructs, clones suitable for agroinfection were constructed. Full-length copies of RNA-1 and RNA-2 were inserted between the sequence of a Cauliflower mosaic virus (CaMV) 35S promoter and a nos terminator and were introduced into the Agrobacterium tumefaciens plasmid, pBINPLUS. Infiltration of leaves of either Nicotiana benthamiana or cowpea (Vigna unguiculata) with a bacterial suspension containing a mixture of the RNA-1- and RNA-2-based plasmids resulted in the plants developing typical CPMV symptoms. To confirm the utility of this approach for use with CPMV-based vectors, a GFP construct based on RNA-2 was adapted for agroinfection. Infiltration of N. benthamiana leaves with a mixture of Agrobacteria containing this construct and the RNA-1 plasmid resulted in high levels of GFP expression. The results demonstrate that agroinfection is a suitable method for the propagation of CPMV-based derivatives.     

Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.     

Phloem specific promoter from a satellite associated with a DNA virus

DNAbeta is a satellite molecule associated with some monopartite begomoviruses and encodes a single gene (betaC1), which is highly conserved in position and size among DNAbeta molecules. A 955 nt fragment of Tomato yellow leaf curl China virus (TYLCCNV) DNAbeta, upstream of the translation start site of betaC1 gene was tested for its promoter activity with gus as a reporter gene. Analysis of beta-glucuronidase (GUS) activity following transient expression assays indicated that the 955 nt fragment had promoter activity and that 3'-deletions of 399 or 173 nt of the fragment resulted in complete loss of its promoter activity. The 5'-deletions of 782 or 556 nt of the fragment, however, did not affect its activity. Histochemical staining revealed that this fragment can be used to express gus gene specifically in phloem tissue of stably transformed tobacco plants. Further studies have indicated that a 173 nt segment from 3'-end of the 955 nt fragment was responsible for basic promoter activity and phloem-specific expression.