Identification and Functional Analysis of Four RNA Silencing Suppressors in Begomovirus Croton Yellow Vein Mosaic Virus

Croton yellow vein mosaic virus (CYVMV), a species in the genus Begomovirus, is a prolific monopartite begomovirus in the Indian sub-continent. CYVMV infects multiple crop plants to cause leaf curl disease. Plants have developed host RNA silencing mechanisms to defend the threat of viruses, including CYVMV. We characterized four RNA silencing suppressors, namely, V2, C2, and C4 encoded by CYVMV and betasatellite-encoded C1 protein (βC1) encoded by the cognate betasatellite, croton yellow vein betasatellite (CroYVMB). Their silencing suppressor functions were verified by the ability of restoring the β-glucuronidase (GUS) activity suppressed by RNA silencing. We showed here for the first time that V2 was capable of self-interacting, as well as interacting with the V1 protein, and could be translocalized to the plasmodesmata in the presence of CYVMV. The knockout of either V2 or V1 impaired the intercellular mobility of CYVMV, indicating their novel coordinated roles in the cell-to-cell movement of the virus. As pathogenicity determinants, each of V2, C2, and C4 could induce typical leaf curl symptoms in Nicotiana benthamiana plants even under transient expression. Interestingly, the transcripts and proteins of all four suppressors could be detected in the systemically infected leaves with no correlation to symptom induction. Overall, our work identifies four silencing suppressors encoded by CYVMV and its cognate betasatellite and reveals their subcellular localizations, interaction behavior, and roles in symptom induction and intercellular virus movement.

Molecular Characterization and Transmission Study of the Begomovirus on Hollyhock Plant Causing Leaf Curl Disease for the First Time in Province Punjab, Pakistan

I. Background: Hollyhock (Alcea rosea) is an ornamental plant belonging to the Malvaceae family and has a remarkable aesthetic and medicinal value. Previously in Pakistan, the hollyhock plant was not found to be infected by begomovirus and the plant first time showed the symptoms of typical leaf curling, puckering as well as thickened veins. II. Methods and Results: During the year 2018, symptomatic samples of the hollyhock plants were collected that exhibited characteristic typical leaf curling, puckering as well as thickened veins. DNA was extracted from the samples and the PCR technique was optimized for the detection of begomovirus followed by sequencing. The samples were detected to be infected with begomovirus by using Av/Ac core, Begomo 01/02, and CLCV 01/02 primer showed positive results with 579bp, 2.8kb, and 1.1kb nucleotide respectively. The betasatellite was amplified by using beta01/02 and CLCuMuBF11/R33 showed positive results with 1400bp and 481bp respectively. Sequencing results showed that diseased hollyhock plants were associated with Cotton leaf curl Multan virus-Rajasthan strain along with Cotton leaf curl Multan betasatellite. III. Conclusion: Hollyhock plants infected by begomovirus has been reported for the first time as a possible source of virus inoculum from Pakistan.

Alterations in the Antioxidant Metabolites in Cotton Leaf Curl Disease Infected and Healthy Cotton Leaves

The upland cotton (Gossypium hirsutum) belongs to the family Malvaceae and cultivated since ancient times in warmer parts of the world. Pakistan is the 4th largest cotton producing country and prominent cotton yarn exporter of the world. Cotton is the major source of natural fiber and significantly contributes to the economy of Pakistan. There are many factors (biotic and abiotic) affecting the cotton yield in Pakistan. The cotton leaf curl disease (CLCuD) is one of the biotic factors and endemic in the most cotton growing areas of the country. The antioxidant enzymes and antioxidant biochemical metabolites play important role during stress. The present study was planned to compare the antioxidant enzymes and metabolites from healthy and CLCuD infected cotton leaves. Antioxidant enzymes activities including catalase, peroxidase and peroxidase were measured through different antioxidant assays and different antioxidant metabolites were also determined. During stress condition antioxidant metabolites serve as signal for the activation of antioxidant enzymes. Present study revealed that antioxidant enzymes were significantly higher in the infected cotton leaves as compared to the healthy cotton leaves. While the amount of antioxidant metabolites like total phenolic contents (TPC) and total flavonoid contents (TFC) were higher in healthy cotton leaves as compared to the infected leaves.

Resilience response to yellow leaf curl disease and identification of resistance gene analogs (RGA) in pepper (Capsicum annuum)

Ayu DK, Maharijaya A, Syukur M, Hidayat SH. 2021. Resilience response to yellow leaf curl disease and identification of resistance gene analogs (RGA) in pepper (Capsicum annuum). Biodiversitas 22: 4731-4739. Pepper yellow leaf curl disease (PYLCD) caused by infection of Begomovirus is a serious threat to pepper production worldwide. Identification of the resistance gene analogs (RGA) and resilience response of pepper against PYLCD is needed especially for selection resistance genotype. Evaluation of resistance response involving 28 pepper genotypes was carried out through transmission of Begomovirus using whitefly (Bemisia tabaci) as vector. The result showed that IPB C12 and F4-012328-6-3 were potential resistance genotypes. A total of 15 R gene analogs (CaRGA) containing NBS motif, namely CARGA1 to CARGA15, were identified by degenerated PCR amplification and database mining. The alignment of deduced amino acid sequence revealed conservation of subdomains Ploop (GKTT), kinase2 (LVVLDDV), RNBSB/kinase3 (IILTTR) and GLPL. BLASTp analysis indicated that 15 RGA showed high homology at deduced amino acid level with R gene identified such as whitefly resistance protein Mi-1.2, Pvr 9 gene for potyvirus, Begomovirus resistance protein, TRGA15 and RGA 13 for putative late blight resistance. Phylogenetic analysis exhibited that isolated sequences distinguished into CNL-NBS groups. These pepper RGA could be considered as candidate sequences of resistance genes.

Evolutionary dynamics of Begomoviruses causing Papaya leaf curl disease in India

The genus begomovirus represents a group of multipartite viruses that significantly damages many agricultural crops, including papaya and drastically influence the overall production. Understanding the genetic variations, mutations and recombination of the begomovirus population infecting papaya has several important implications in alleviating substantial losses, mainly in developing countries, including India. In an attempt for a bioinformatics driven study of diversity and variability of papaya leaf curl disease in India, a total of thirty-two (32) DNA-A and sixteen (16) betasatellite sequences were retrieved from GenBank. An uneven distribution of evolutionary divergence has been observed across the branch length, which triggered the estimated recombinational event. Interestingly, a maximum of the begomoviruses were found to be intra-species recombinants. Further genetic variability, selection pressure, and substitution rate acting on the population were estimated and found to be high enough to support the evolution of geminiviruses. Genetic divergence composition in all begomovirus datasets revealed predominance of nucleotide diversity driven by mutation. The analysis indicates that even though a significant fraction of the genetic variations might be due to recombination but, it was constantly lower than the mutation rate. Thus, the diversification of the begomovirus population is principally impelled by mutational dynamics.