Research On Disease Resistance Function Of VqJAZ7 From Chinese Wild Vitis Quinquangularis

JAZ family proteins comprise a class of transcriptional repressors that silence jasmonate-inducible genes.Although a considerable amount of research has been carried out on this gene family,there is still very little information available on the role of specific JAZ gene members in multiple pathogen resistance.In this study,we investigated the potential resistance function of the VqJAZ7 gene from a disease-resistant wild grapevine,Vitis quinquangularis clone“Shang-24”,through heterologous expression in Arabidopsis thaliana against the biotrophic fungus powdery mildew,necrotrophic fungus Botrytis cinerea,and semi-biotrophic bacteria Pseudomonas syringae pv.tomato DC3000.1.When we investigated the biotrophic fungal disease Powdery mildew,fewer disease symptoms were observed in transgenic lines compared with nontransgenic controls at 7 dpi when we inoculated the nontransgenic control plants and VqJAZ7 overexpressing plants.This result was further supported by trypan blue,NBT and DAB staining which indicated higher level of cell death,more superoxide anions?O₂??,and H₂O₂ accumulation respectively,in transgenic lines.This histochemical staining result further suggested that transgenic lines resisted G.cichoracearum infection by causing more cell death,which is a limiting factor to biotrophic pathogens.We observed that AtICS1 and AtPR1,the SA-dependent disease resistance genes,were significantly upregulated in transgenic plants after PM infection.This indicates that JAZ7 may contribute to resistance to powdery mildew by promoting expression of SA-mediated genes.On the other hand,AtLOX3 and AtPDF1.2were expressed to lower levels after PM infection,suggesting suppression of JA signaling.2.In contrast,in the case of B.cinerea,fewer disease symptoms were observed on nontransgenic control plants than on transgenic lines expressing VqJAZ7 at 72 hpi when we inoculated the nontransgenic control plants and VqJAZ7 overexpressing plants.We also found significantly larger lesions on transgenic lines as compared to WT plants as previous studies have demonstrated that necrotrophic fungi induced PCD.We also measured the transcript levels of defense-related genes,including AtPR1,AtICS1,AtPDF1.2 and AtLOX3in response to B.cinerea at 0,24,48,and 72 hpi,to gain understanding of the pathways by which resistance may be mediated.After inoculation with B.cinerea,AtICS1 was expressed to⁹.9-fold and 7-fold higher in VqJAZ7 transgenic lines compared to control plants at 48and 72 hpi respectively.Likewise,the expression of AtPR1 was elevated 4.4-fold and 4.1-fold at 48 and 72 hpi respectively in the transgenic plants.In contrast,AtPDF1.2 and AtLOX3 were expressed to lower levels in transgenic lines as compared to controls at all time points post-infection.AtPDF1.2 and AtLOX3 were both expressed differentially,but this expression was lower than in nontransgenic control plants.Active PCD promotes the infection of B.cinerea in the host.In case of a necrotrophic pathogen like B.cinerea,ROS exhibited susceptibility in the host plant and its accumulation at later time points directly benefited the fungus.We also noted more cell death and higher levels of H₂O₂,in transgenic lines compared with control plants,indicating that B.cinerea induced cell death and produced more H₂O₂.We also noted that the activities of antioxidant enzymes were lower in the VqJAZ7 transgenic lines than in the nontransgenic control lines,which further indicated that heterologous expression of VqJAZ7 promoted B.cinerea disease progression.3.VqJAZ7 transgenic plants were also less resistant to infection by Pst DC3000 when we tested the transgenic lines along with non-transgenic control WT plants.Histochemical staining with trypan blue and NBT,indicated high levels of cell death and accumulation of more superoxide anions in WT plants as compared to VqJAZ7 transgenic lines when infected with Pst DC3000.In our study both SA-and JA-dependent genes were not induced after P.syringae infection.Coronatine production by P.syringae might not only suppress SA-mediated gene expression,but also repress expression of JA-mediated genes due to the structural and functional similarity between coronatine and JA-Ile.To further validate our results,we also challenged the VqJAZ7 transgenic plants with defense-response elicitors including the peptide flg22,and lipopolysaccharide,which serves as a stimulant of innate immunity.We found that the transgenic plants accumulated less callose than controls after injection of Pst DC3000,LPS and flg22 which further validate that overexpression VqJAZ7resulted in susceptibility to this pathogen.