Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus

Vegetables are an important source of income and high-value crops for small farmers. Chilli (Capsicum spp.) is one of the most economically important vegetables of Pakistan and it is grown throughout the country. It is a rich source of nutrition especially vitamins A, B, C and E along with minerals as folic acid, manganese (Mn), potassium (K) and molybdenum (Mo). Chilli possesses seven times more amount of vitamin C than an orange. Vitamin A, C and beta-carotenoids are strong antioxidants to scavenge the free radicals. Chilli production is restricted due to various biotic factors. Among these viruses, Chilli veinal mottle virus (ChiVMV) is one of the most destructive and menacing agents that inflicts heavy and colossal losses that accounted for 50% yield loss both in quality and quantity. Pathogen-Derived Resistance (PDR) approach is considered one of the effective approaches to manage plant viruses. In this study, ChiVMV was characterized on a molecular level, the coat protein (CP) gene of the virus was stably transformed into Nicotiana benthamiana plants using Agrobacterium tumefaciens. The transgenic plants were challenged with the virus to evaluate the level of resistance of plants against the virus. It was observed that the plants expressing CP gene have partial resistance against the virus in terms of symptoms’ development and virus accumulation. Translation of this technique into elite chilli varieties will be resulted to mitigate the ChiVMV in the crop as well as an economic benefit to the farmers.

Identification and Characterization of a Novel Potyvirus Infecting Paris Yunnanensis

The complete genomic sequence of a novel potyvirus from Paris yunnanensis was determined by high-throughput sequencing then confirmed by Sanger sequencing. Its genomic RNA consists 9600 nucleotides (nt) excluding the 3’-terminal poly (A) tail, containing a typical large open reading frame (ORF) of potyviruses and encoding a putative polyprotein of 3098 amino acids (aa). Pairwise comparison analysis showed the virus shares sequence identity with other members of Potyvirus was 53.0% to 57.8% at genome sequence level, and 39.3% to 51.2% at polyprotein sequence level. Phylogenetic analysis indicated that the virus was most closely related to the subgroup of plum pox virus and that of chilli veinal mottle virus within the genus Potyvirus. These results suggest that the virus should be considered as a distinct species within the genus Potyvirus and was tentatively named as “Paris mottle associated virus” (PMaV).

Identification of Viruses Infecting Cigar and Flue-cured Tobacco Using Next Generation Sequencing and Establishment of Multiplex RT-PCR

Early, precise and simultaneous identification of the plant viruses is of great significance on preventing the spread of the viruses as well as reducing losses on agricultural yield. In this study, identification of plant viruses from symptomatic samples collected from cigar tobacco planting area in Deyang and flue-cured tobacco planting area in Luzhou city of Sichuan Province China was conducted by the deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform and plant virus specific contigs were generated based on the virus derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis was performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples with a total of 105 contigs being mapped to the closest plant viruses with the length range from 34~1720 nt. The results indicated that the major viruses were potato virus Y (PVY), Chilli veinal mottle virus (ChiVMV), tobacco vein banding mosaic virus (TVBMV), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. These results provide theoretical basis and convenient methods for rapid detection and control of viruses on cigar and flue-cured tobacco.

Molecular Characterization of Two Isolates of Wild Tomato Mosaic Virus and Chilli Veinal Mottle Virus Co-infecting Chilli Pepper in China

The present study reports observation of a field chilli pepper disease consisting of a co-infection with two potyviruses: Wild tomato mosaic virus Dehong isolate (WTMV-Dh) and Chili veinal mottle virus Dehong isolate (ChiVMV-Dh). We obtained the complete genome sequences of these two viruses by NGS sequencing. The WTMV-Dh is 9,598 nucleotides (nt) in length and encodes a complete polyprotein of 3,075 amino acids (aa). The polyprotein of WTMV-Dh shares 76.1–82.6% nt and 85.3–89.5% aa identities with the other three WTMV isolates reported previously. The ChiVMV-Dh is 9688 nt in length and encodes a complete polyprotein with 3, 089 aa. The polyprotein of ChiVMV-Dh shares 80.8–92.2% nt and 85.3–95.6% aa identities with the other ChiVMV isolates reported previously. Following phylogenetic analysis based on the polyprotein sequences of other potyviruses, WTMV-Dh clustered with the Vietnam strain WTMV-Laichau while ChiVMV-Dh clustered with several ChiVMV Sichuan isolates. Evaluation of the recombination events within the WTMV and ChiVMV subgroups indicated that some putative recombination events occurred in critical regions. These regions include the N-terminal of HC-Pro and P1 region of WTMV-Dh, CP and the P3 to CI region of ChiVMV-Dh, which may be new evidence of adaptive evolution of potyviruses.

Identification of Two New Isolates of Chilli veinal mottle virus From Different Regions in China: Molecular Diversity, Phylogenetic and Recombination Analysis

Chilli veinal mottle virus (ChiVMV) is an important plant pathogen with a wide host range, causing serious yield losses in pepper production all over the world. Recombination is a major evolutionary event for single-stranded RNA viruses, which helps isolates adapt to new environmental conditions and hosts. Recombination events have been identified in multiple potyviruses, but so far, there have been no reports of recombination events among the ChiVMV population. We here detected ChiVMV in pepper samples collected from Guangxi and Yunnan provinces for the first time and amplified the nearly full-length sequences. Phylogenetic and recombination analysis were performed using the new sequences and the 14 full-length and 23 capsid protein (CP) sequences available in GenBank. Isolates tend to cluster on a geographical basis, indicating that geographic-driven evolution may be an important determinant of ChiVMV genetic differences. A total of 10 recombination events were detected among the ChiVMV sequences using RDP4 with a strict algorithm, and both the Guangxi and Yunnan isolates were identified as recombinants. Recombination appears to be a significant factor affecting the diversity of ChiVMV isolates.

Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

Plant symptoms are derived from specific interactions between virus and host components. However, little is known about viral or host factors that participate in the establishment of systemic necrosis. Here, we showed that helper component proteinase (HCPro), encoded by Chilli veinal mottle virus (ChiVMV), could directly interact with catalase 1 (CAT1) and catalase 3 (CAT3) in the cytoplasm of tobacco (Nicotiana tabacum) plants to facilitate viral infection. In vitro, the activities of CAT1 and CAT3 were inhibited by the interaction between HCPro and CATs. The C-terminus of HCPro was essential for their interaction and was also required for the decrease of enzyme activities. Interestingly, the mRNA and protein level of CATs were up-regulated in tobacco plants in response to ChiVMV infection. Nicotiana tabacum plants with HCPro overexpression or CAT1 knockout were more susceptible to ChiVMV infection, which was similar to the case of H2O2-pre-treated plants, and the overexpression of CAT1 inhibited ChiVMV accumulation. Also, neither CAT1 nor CAT3 could affect the RNA silencing suppression (RSS) activity of HCPro. Our results showed that the interaction between HCPro and CATs promoted the development of plant systemic necrosis, revealing a novel role for HCPro in virus infection and pathogenicity.