Gene function studies of begomovirus genes expressed from PVX vector in Nicotiana benthamiana

Abstract

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases of crops are caused by begomviruses, particularly in developing countries. These include African cassava mosaic virus (ACMV), which causes significant losses to the subsistence crop cassava in sub-Saharan Africa, and the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to the mainstay of the economy of Pakistan, cotton. The study presented here was designed to provide us with a better understanding of begomovirus pathogenicity, virus host interactions and the roles of individual virus-encoded genes in these interactions, as well as the effects of environmental factors on pathogenicity. Ultimately the information gained may be useful in allowing the design of better control strategies using either natural or engineered resistance. To investigate, on a wider scale than has previously been conducted, which gene products encoded by begomoviruses are involved in symptom induction/pathogenicity, all genes encoded by ACMV, Cotton leaf curl Multan virus (CLCuMV)/Cotton leaf curl Multan betasatellite (CLCuB), Cabbage leaf curl virus (CbLCuV) and Tomato yellow leaf curl virus (TYLCV), representing four different classes of begomoviruses, were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. All efforts to transform the PVX construct with the TYLCV replication associated protein gene (rep) into Agrobacterium were unsuccessful, indicating that this Rep may be toxic to Agrobacterium and precluding its use in all further studies. With the exception of the REn proteins, the TrAP proteins of ACMV and CLCuMV, the CPs of CLCuMV and CbLCuV, and the DNA B encoded MP and NSP of CbLCuV and MSP of ACMV, all other begomovirus proteins, when expressed from PVX, induced a phenotype above and beyond the mild symptoms induced by PVX in N. benthamiana. All three Rep proteins induced a severe symptom phenotype, however, for CLCuMV and CbLCuV, after the initial severe symptoms plants gradually recovered. Although all (A)V2 proteins induced severe symptoms, that of CLCuMV additionally induced a necrotic response in both inoculated leaves and leaves developing subsequent to inoculation that was reminiscent of a hypersensitive response. PVX-mediated expression of CLCuMB βC1 induced the most unusual symptoms in N. benthamiana. These symptoms resembled those induced by CLCuD in cotton and consisted of leaf curling, vein yellowing, stunting and the formation of enations on the undersides of leaves. These results indicate that, even for quite closely related viruses within a single genus, each virus interacts with plants (or at least N. iiibenthamiana) in a distinct fashion and that the ultimate outcome (the visible symptoms) are likely a complex interaction of multiple virus-encoded genes with distinct host factors, or affecting the same host factors to varying degrees. Overall it is clear that the findings with one virus do not set a precedent for the other viruses. MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene expression in plants and animals. miRNAs are involved in a variety of activities, including plant development, signal transduction, protein degradation, response to environmental stress, and pathogen invasion. Several studied have shown that miRNA-directed processes might be a general feature of virus pathogenicity. In order to study this phenomenon, in relation to begomoviruses, infections of N. benthamiana by four distinct virus species were assessed for their effects on ten miRNAs known to be important in plant development. Additionally, the effects of the expression of all genes encoded by the four begomoviruses from a PVX vector on miRNA levels were studied. Northern blot analysis using specific oligonucleotide probes for miRNAs showed that, in general, begomovirus infection increases the accumulation of miRNAs. However, there was no general consistency between the viruses, each affecting different miRNAs and to varying extents. The analysis showed that, essentially, all begomovirus-encoded gene products have the ability to influence host miRNA levels, the first time this has been demonstrated. It was also found that genes encoding suppressor of gene silencing affect the miRNA level in a significant way when compared with non suppressors. Again there was a lack of consistency, each virus appearing to use a different protein, or proteins, to influence miRNA levels, although there were some minor trends apparent such as, for example, the TrAP protein of all viruses analyzed being the strongest up-regulator of the widest range of miRNAs. Furthermore, the results suggest that miRNA binding by begomovirus suppressors such as TrAP and (A)C4, and by implication also those of other phytopathogenic viruses, are collateral damage; thus that they are unintentional and result merely from the overlap of the siRNA and miRNA pathways. RNA interference (RNAi) is a natural defense response of plants against invading viruses. As a counter-defense viruses encode suppressors of gene silencing that allow them to effectively invade plant hosts. Using a novel quantitative real time PCR (qPCR) assay and conventional northern blot analysis, the ability of all genes encoded by the begomovirus CLCuMV and its associated betasatellite, CLCuMB (which together cause CLCuD), were assessed for their ability to suppress RNAi. The analysis showed that the V2, TrAP, C4 and βC1 proteins exhibit suppressor activity. Although each of these proteins has, for other viruses, been previously shown to have suppressor activity, this is ivthe first time all proteins encoded by a geminivirus (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified. Since all the proteins were examined in a single experiment, this also allowed comparison of the relative suppressor activity of each protein, which showed that the strongest activity was for V2. Environmental conditions have a marked effect on the infectivity and spread of viruses in plants. To investigate the effects of temperature and light intensity on virus spread and gene silencing in plants, the CLCuMV-encoded V2 protein and the green fluorescent protein (GFP) were used as markers of PVX infection. Both light and temperature were found to have profound effects on PVX infection, with lower light and lower temperatures enhancing virus symptoms and gene silencing. The enhanced symptoms and silencing were associated with higher levels of viral RNA and GFP- specific siRNA. Thus siRNA accumulation and viral transcript accumulation has a positive correlation. The presence of high levels of siRNA under low temperature and low light also showed that the RNA silencing pathway is more active under these conditions. These findings suggest that RNA silencing has no role in the differential behavior of PVX in response to varying environmental conditions.