Molecular Mechanism Of Hypersensitive Response Induced By RepA Protein Encoded By Oat Dwarf Viurs On Tobacco Plants

Incompatible interaction between virus and plant often leads to rapid cell death and a limited necrosis, which is called hypersensitive response (HR). Oat dwarf virus (ODV) is a member of the Mastrevirus genus in the Geminiviridae family and its genome is a single stranded circular DNA. ODV encodes four proteins (V1, V2, Rep and RepA) in virion-sense strand and complementary-sense strand. In this study, we have investigated a HR-like response elicited in tobacco plants by inoculation with ODV infectious clone and have found that only expression of the ODV RepA protein, but not other ODV-encoded proteins, elicits the HR-like response associated with a burst of H2O2in Nicotiana benthamiana and a high expression of HSR203J and H1N1served as marker genes of HR. Deletion mutagenesis indicated that the first9amino acids (aa) at the N-terminus of RepA have an important role in HR induction, and that two regions located between aa residues173and195or sited between aa residues241and260near the C-terminus are essential for HR elicitation. Confocal microscopy and electron microscopy visualization showed the RepA protein is localized in the nuclei of plant cells and might contain bipartite nuclear localization signals. The HR-like lesions mediated by RepA is inhibited in tobacco plants by temperatures above30℃. We also confirmed that HR induced by RepA is regulated by JA signaling molecule using gain-and loss-of-function experiments. Pretreatment with methyl jasmonate (MeJA) or VIGS silencing of COI (Coronatine-Insensitive1) and AOS (allene oxide synthase), the key components of the JA-defense pathway, could significantly promote or delay HR induced by RepA. To our knowledge, this is the first report of an elicitor of HR of mastreviruses.To determine the molecular mechanism of HR induced by RepA, we used yeast two hybrid system to screen cDNA of N. benthamiana with RepA mutant deleted the transcriptional domain as a bait protein and discovered S-adenosylmethionine decarboxylase1(NbSAMDC1) could interact with RepA protein. Furthermore, BiFC and GST pull down asaay showed that NbSAMDC1and RepA protein interacts in vivo and vitro. NbSAMDC1is a key enzyme in polyamine biosynthesis. We found that the expression of related gene involved in polyamine biosynthesis pathway including NbSAMDC1can be induced by transient expression of RepA protein or inoculation of ODV infectious clone when tested by qRT-PCR technology. On the other hand, Methylglyoxal bis-amidinohydrazone (MGBG), a competitive inhibitor of SAMDC. treatment significantly inhibited HR induced by RepA. The aboved results suggested that the polyamine is likely invoved in RepA-induced HR as a signal molecular.In order to further understand the biological meaning of interaction between NbSAMDC1and RepA protein, we used TRV or PVX vector to silence or overexpress NbSAMDC1. The results showed that HR induced by RepA delayed in NbSAMDCl-silenced plants and HR appeared ealier in NbSAMDC1overexpression plants. Further study on the NbSAMDC1overexpressed transgenic plants or NbSAMDC1-silenced plants using RNAi technology showed the same results as aboved.We also analyzed the expression profiles of RepA-induced HR using Illumina sequencing platform, HiSeq2000. There was a total of4898up-regulated DEGs (significant differentially expressed genes, DEGs) and8486down-regulated DEGs distributed in125pathways of KEGG database. We also found most of the genes involved in photosynthesis were significantly inhibited. A gene named NbFDN1(ferredoxin1), was choosen for further study. This gene has been reported to interact with Tsip1and involve in the regulation of biotic and abiotic stresses. Our study showed that the two genes involved in the regulation of HR induced by RepA and expression of RepA in leaf cells caused the thylakoid degradated, with the expression of autophagy related genes reduced generally, suggesting that the expression of ODV RepA could interfer autophagy to affect the degradation of chloroplast and regulate plant defense response.