ATG8f Interacts with Chilli Veinal Mottle Virus 6K2 Protein to Limit Virus Infection.

Autophagy, as a conserved protein degradation pathway in plants, has also been reported to be intricately associated with antiviral defense mechanisms. However, the relationship between chilli veinal mottle virus (ChiVMV) and autophagy has not been investigated in the existing research. Here, we reveal that ChiVMV infection caused the accumulation of autophagosomes in infected Nicotiana benthamiana leaves and the upregulation of autophagy-related genes (ATGs). Moreover, the changes in gene expression were correlated with the development of symptoms. Treatment with autophagy inhibitors (3-MA or E-64D) could increase the infection sites and facilitate virus infection, whereas treatment with the autophagy activator (Rapamycin) limited virus infection. Then, ATG8f was identified to interact with ChiVMV 6K2 protein directly in vitro and in vivo. The silencing of ATG8f promoted virus infection, whereas the overexpression of ATG8f inhibited virus infection. Furthermore, the expression of 6K2-GFP in ATG8f- or ATG7-silenced plants was significantly higher than that in control plants. Rapamycin treatment reduced the accumulation of 6K2-GFP in plant cells, whereas treatment with the inhibitor of the ubiquitin pathway (MG132), 3-MA, or E-64D displayed little impact on the accumulation of 6K2-GFP. Thus, our results demonstrated that ATG8f interacts with the ChiVMV 6K2 protein, promoting the degradation of 6K2 through the autophagy pathway.

Glucose-6-phosphate dehydrogenase promotes the infection of Chilli veinal mottle virus through affecting ROS signaling in Nicotiana benthamiana.

MAIN CONCLUSION: G6PDH negatively regulates viral accumulation in Nicotiana benthamiana through RBOHB-associated ROS signaling. Anti-oxidative metabolism and phytohormone-mediated immunity responses play important roles in virus infection. Glucose-6-phosphate dehydrogenase (G6PDH) is an enzyme in the pentose phosphate pathway, which plays an important role in maintaining intracellular redox homeostasis and has functions in plant growth, development and stress tolerance. However, the role of G6PDH in plants response to virus infection is poorly understood. In this study, NbG6PDH was found to be down-regulated after Chilli veinal mottle virus (ChiVMV-GFP) infection in Nicotiana benthamiana. Subcellular localization of NbG6PDH showed that it was punctate distributed in the protoplasm. Silencing of NbG6PDH reduced the sensitivity of N. benthamiana plants to ChiVMV-GFP. By contrast, transient overexpression of NbG6PDH promoted the accumulation of the virus. The results of physiological indexes showed that glutathione (GSH), catalase (CAT) and proline played an important role in maintaining plants physiological homeostasis. The results of gene expression detection showed that jasmonic acid/ethylene (JA/ET) signaling pathway was significantly correlated with the response of N. benthamiana to ChiVMV-GFP infection, and the changes of N. benthamiana respiratory burst oxidase homologues B (NbRBOHB) indicated that the NbG6PDH-dependent ROS may be regulated by NbRBOHB. Pretreatment of the inducer of reactive oxygen species (ROS) promoted virus infection, whereas inhibitor of ROS alleviated virus infection. Thus, our results indicate that the promoting effect of NbG6PDH on ChiVMV-GFP infection may be related to the NbRBOHB-regulated ROS production.

Construction of full-length cDNA infectious clones of Chilli veinal mottle virus.

Chilli veinal mottle virus (ChiVMV), a member of the genus Potyvirus in the family Potyviridae, causes severe diseases and poses a great threat to solanaceous crops. Reverse genetics technology is an efficient tool to facilitate the study of virus biology and pathogenicity. However, the construction of an infectious cDNA clone of ChiVMV is yet to be reported. In this study, full-length cDNA infectious clones of ChiVMV and GFP-tagged ChiVMV were constructed using yeast homologous recombination for the first time. These infectious clones were able to successfully infect host plants (Nicotiana benthamiana, Nicotiana tabacum and Solanum lycopersicum) by Agrobacterium-mediated infiltration and cause vein banding and leaf curling symptoms. Mutations were introduced to pChiVMV-GFP to investigate the role of key amino acids in ChiVMV 6K2. The results showed that substitution mutants of leucine (L9, 11) to alanine acid (A), tryptophan (W15) to alanine acid (A), and glycine (G29, 33) to valine acid (V) reduced the viral accumulation and the mutant clones were unable to induce the symptoms in N. benthamiana plants. Taken together, these infectious clones we developed will be effective tools for future studies of the function of viral factors encoded by ChiVMV and the interactions between ChiVMV and its different host plants.

Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus / Avaliação do gene CP do vírus Chilli veinal mottle que expressa plantas transgênicas Nicotiana benthamiana quanto à resistência a doenças contra o vírus

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 betacarotenoids 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.

Vegetais são uma importante fonte de renda e culturas de alto valor para os pequenos agricultores. A pimenta-malagueta (Capsicum spp.) é uma das hortaliças mais importantes economicamente do Paquistão e é cultivada em todo o país. É uma rica fonte de nutrição, especialmente vitaminas A, B, C e E com minerais como ácido fólico, manganês (Mn), potássio (K) e molibdênio (Mo). O pimentão possui sete vezes mais vitamina C do que a laranja. Vitaminas A e C e betacarotenoides são antioxidantes fortes para eliminar os radicais livres. A produção de pimenta é restrita devido a vários fatores bióticos. Entre esses vírus, o ChiVMV é o agente mais destrutivo e ameaçador que inflige perdas pesadas e colossais que representam 50% da perda de rendimento, tanto em qualidade quanto em quantidade. A abordagem de resistência derivada de patógenos (PDR) é considerada uma das abordagens eficazes para gerenciar os vírus de plantas. Neste estudo, ChiVMV foi caracterizado em nível molecular e o gene CP do vírus foi transformado de forma estável em plantas Nicotiana benthamiana usando Agrobacterium tumefaciens. As plantas transgênicas foram desafiadas com o vírus para avaliar seu nível de resistência contra o vírus. Observou-se que as plantas que expressam o gene CP apresentam resistência parcial ao vírus em termos de desenvolvimento de sintomas e acúmulo de vírus. A tradução dessa técnica em variedades de pimenta de elite resultará na mitigação do ChiVMV na safra, bem como em benefícios econômicos para os agricultores em termos de melhor rendimento e baixo custo de produção.

Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus / Avaliação do gene CP do vírus Chilli veinal mottle que expressa plantas transgênicas Nicotiana benthamiana quanto à resistência a doenças contra o vírus

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.

Vegetais são uma importante fonte de renda e culturas de alto valor para os pequenos agricultores. A pimenta-malagueta (Capsicum spp.) é uma das hortaliças mais importantes economicamente do Paquistão e é cultivada em todo o país. É uma rica fonte de nutrição, especialmente vitaminas A, B, C e E com minerais como ácido fólico, manganês (Mn), potássio (K) e molibdênio (Mo). O pimentão possui sete vezes mais vitamina C do que a laranja. Vitaminas A e C e betacarotenoides são antioxidantes fortes para eliminar os radicais livres. A produção de pimenta é restrita devido a vários fatores bióticos. Entre esses vírus, o ChiVMV é o agente mais destrutivo e ameaçador que inflige perdas pesadas e colossais que representam 50% da perda de rendimento, tanto em qualidade quanto em quantidade. A abordagem de resistência derivada de patógenos (PDR) é considerada uma das abordagens eficazes para gerenciar os vírus de plantas. Neste estudo, ChiVMV foi caracterizado em nível molecular e o gene CP do vírus foi transformado de forma estável em plantas Nicotiana benthamiana usando Agrobacterium tumefaciens. As plantas transgênicas foram desafiadas com o vírus para avaliar seu nível de resistência contra o vírus. Observou-se que as plantas que expressam o gene CP apresentam resistência parcial ao vírus em termos de desenvolvimento de sintomas e acúmulo de vírus. A tradução dessa técnica em variedades de pimenta de elite resultará na mitigação do ChiVMV na safra, bem como em benefícios econômicos para os agricultores em termos de melhor rendimento e baixo custo de produção.

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

Abstract 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.

Resumo Vegetais são uma importante fonte de renda e culturas de alto valor para os pequenos agricultores. A pimenta-malagueta (Capsicum spp.) é uma das hortaliças mais importantes economicamente do Paquistão e é cultivada em todo o país. É uma rica fonte de nutrição, especialmente vitaminas A, B, C e E com minerais como ácido fólico, manganês (Mn), potássio (K) e molibdênio (Mo). O pimentão possui sete vezes mais vitamina C do que a laranja. Vitaminas A e C e betacarotenoides são antioxidantes fortes para eliminar os radicais livres. A produção de pimenta é restrita devido a vários fatores bióticos. Entre esses vírus, o ChiVMV é o agente mais destrutivo e ameaçador que inflige perdas pesadas e colossais que representam 50% da perda de rendimento, tanto em qualidade quanto em quantidade. A abordagem de resistência derivada de patógenos (PDR) é considerada uma das abordagens eficazes para gerenciar os vírus de plantas. Neste estudo, ChiVMV foi caracterizado em nível molecular e o gene CP do vírus foi transformado de forma estável em plantas Nicotiana benthamiana usando Agrobacterium tumefaciens. As plantas transgênicas foram desafiadas com o vírus para avaliar seu nível de resistência contra o vírus. Observou-se que as plantas que expressam o gene CP apresentam resistência parcial ao vírus em termos de desenvolvimento de sintomas e acúmulo de vírus. A tradução dessa técnica em variedades de pimenta de elite resultará na mitigação do ChiVMV na safra, bem como em benefícios econômicos para os agricultores em termos de melhor rendimento e baixo custo de produção.

Characterization and a RT-RPA assay for rapid detection of Chilli Veinal mottle virus (ChiVMV) in tobacco.

BACKGROUND: Chilli veinal mottle virus (ChiVMV), which belongs to the genus Potyvirus of the family Potyviridae, mainly infects solanaceous plants and has caused serious economic losses in Asia and Africa. Tobacco plants infected with ChiVMV suffered from punctate necrosis of leaves, leaf deformation, systemic necrosis of leaves and stems, and eventually plant death. However, ChiVMV infection could not usually be identified given the lack of rapid and efficient detection assays in tobacco plants. Therefore, an isolate of tobacco-infecting ChiVMV (ChiVMV-LZ) was obtained, and a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification (RT-RPA), was established to detect ChiVMV in tobacco plants. METHODS: In this study, the full-length genome of ChiVMV-LZ was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) assays. The genome sequence of ChiVMV-LZ was characterized by sequence alignment and phylogenetic analysis. Then, a RT-RPA assay was established for rapid and sensitive detection of ChiVMV-LZ in tobacco. Additionally, the established RT-RPA assay was compared to the RT-PCR assay in aspect of sensitivity and application in field-collected tobacco samples. RESULTS: ChiVMV-LZ was isolated from diseased tobacco in Luzhou, Sichuan, China. The tobacco plants inoculated with ChiVMV-LZ showed typical symptoms of yellow and round spots on the leaves, and curled and folded leaf margin, similar to those observed on naturally ChiVMV-infected tobacco in the field. The full-length genomic sequence of ChiVMV-LZ was determined to be 9742 nucleotides. Sequence alignment and phylogenetic analysis showed that ChiVMV-LZ was most closely related to ChiVMV-Yp8 isolated from pepper plants in Sichuan province while distantly related to ChiVMV-YN from tobacco in Yunnan province, indicating a possibly geographical differentiation of ChiVMV isolates. Additionally, a RT-RPA assay was established for rapid detection of ChiVMV in tobacco. The RT-RPA has no cross-reaction with other related tobacco viruses and is about 10-fold more sensitive than conventional RT-PCR method. CONCLUSION: The characterization of ChiVMV-LZ infecting tobacco was determined, and the established RT-RPA assay provides a reliable and effective method for rapid detection of ChiVMV in tobacco.

N gene enhances resistance to Chilli veinal mottle virus and hypersensitivity to salt stress in tobacco.

Plants use multiple mechanisms to fight against pathogen infection. One of the major mechanisms involves the disease resistance (R) gene, which specifically mediates plant defense. Recent studies have shown that R genes have broad spectrum effects in response to various stresses. N gene is the resistance gene specifically resistant to Tobacco mosaic virus (TMV). However, the role of N gene in abiotic stress and other viral responses remains obscure. In this study, we investigated the mechanisms by which N regulates plant defense responses under Chilli veinal mottle virus (ChiVMV) infection and salt stress. Here, we monitored the physiological and molecular changes of tobacco plants under virus attack. The results showed that when tobaccoNN and tobacconn plants were exposed to ChiVMV, tobaccoNN plants displayed higher susceptibility at five days post infection (dpi), while tobacconn plants exhibited higher susceptibility at 20 dpi. In addition, accumulation of reactive oxygen species (ROS) and expression of HARPIN-INDUCED1(NtHIN1) were higher in tobaccoNN plants than in tobacconn plants at 5 dpi. Interestingly, the pathogenesis-related gene (NtPR1 and NtPR5), the activities of antioxidant enzymes, and the content of salicylic acid (SA) in tobaccoNN plants increased compared with those in tobacconn plants. It was suggested that the N gene induced a hypersensitive response (HR) and enhanced the systemic resistance of plants in response to ChiVMV via the SA-dependent signaling pathway. In addition, the N gene was also induced significantly by salt stress. However, tobaccoNN plants showed hypersensitivity toward increased salt stress, and this hypersensitivity was dependent on abscisic acid and jasmonic acid but not SA. Taken together, our results indicate that the N gene appears to be important in the plant response to ChiVMV infection and salt stress.