Molecular Mechanisms Of Cf-dependent ETI And Xoo-induced Nonhost Resistance In Nicotiana Benthamiana

Plant disease resistance is classified as host resistance and nonhost resistance depending on whether the involved plant is the host of the involved pathogen. The two types of resistance share many similar mechanisms yet some obvious differences exist for them. The host resistance contains two layers namely pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI). The tomato-Cladosporium fulvum pathosystem is a typical gene-for-gene resistance interaction system. Tomato Cf genes conferred ETI to the fungus carrying the complementary Avr. Xanthomonas oryzae pv. oryzae (Xoo), causing rice bacterial blight, with a narrow host range, causes strong hypersensitive response (HR) and resistance in its nonhost Nicotiana benthamiana. This thesis research was conducted under the background of the two issues. Firstly, it is traditionally thought that the resistance (R) genes, which confer ETI, are not regulated for its expression at transcriptional or post transcriptional level. In this research, we reveal that expression of Cf genes is regulated at both transcriptional and post transcriptional levels. Additionally, the mechanisms underlying the Zoo-induced HR and nonhost resistance in N. benthamiana are largely unknown. In this study, several strategies are employed to screen the genes involved in regulating this HR and nonhost resistance. In addition, the role and mechanisms of Agrobacterium in inhibition of this HR and nonhost resistance are elucidated. The main results are as follows:1. The mechanisms underlying expression of tomato leaf mould resistance genes Cf-4and Cf-9were revealed. Mechanisms to regulate expression of Cf-4and Cf-9genes were analyzed at transcriptional level (DNA methylation) and post transcriptional level (miRNA). At transcriptional level, high temperature repressed while Avr strongly induced the expression of Cf genes. In addition, expression of Cf-4enhanced while that of Cf-9was not affected by the age increment. DNA methylation of promoter was involved in regulation of both high temperature-suppressive Cf-9expression and Avr9-induced Cf-9expression at normal temperature, as well as Avr-induced Cf expression during temperature shift from high to normal. Employing VIGS technique, effect of tomato methyltransferase and demethyltransferase genes on Cf gene expression was analyzed. The results showed that silencing of some methyltransferase and all4demethyltransferase genes significantly altered Cf gene expression, indicating that these genes might contribute to regulation of Cf gene expression. At post transcriptional level, a21nt miRNA targeting both Cf-4and Cf-9mRNAs was predicted and experimentally verified by detecting the effective degradation of Cf transcripts when co-expressed both target and pre-miRNA of the miRNA.2. A set of genes involved in regulating the Cf-4/Avr4-dependent HR were screened out. Through analyzing the function of genes encoding differentially expressed proteins as well as cDNA library VIGS screening, we obtained28genes involved in regulating the Cf-4/Avr4-dependent HR from over500VIGS constructs. Among which were positive HR regulators including a ribonucleoprotein, a DNA binding protein and a Dof zinc finger protein DOF3.3. The remaining22genes served as negative regulator of the Cf-4/Avr4-dependent HR, such as BTB/POZ and TAZ domain-containing protein1, Ring finger protein26, CP-interacting protein3, SUMO-conjugating enzyme SCE1and red chlorophyll catabolite reductase.3. The N. benthamiana-Xoo nonhost resistance system was established and optimized, and the indispensible role of H2O2accumulation in the Xoo-induced HR and nonhost resistance was revealed. We established that infiltration with1×108cfu/ml Xoo inocula in fully expanded leaves and grown at28℃resulted in the most rapid and strong HR and resistance. Xoo inoculation caused high level of H2O2accumulation, which led to strong HR and high expression of PR and HR marker genes, and finally dramatic reduce of Xoo bacterial number, manifesting nonhost resistance. Experiments of removal of H2O2by catalase demonstrated that H2O2accumulation is pivatol in the Jtbo-induced HR and nonhost resistance.4. A set of genes involved in regulating the Xoo-induced nonhost resistance were screened out. By analyzing the function of genes encoding differentially expressed proteins and cDNA library VIGS screening, we obtained8genes that were involved in regulating the Xoo-induced nonhost resistance from over500VIGS constructs. Among which were three negative regulators including a carbonic anhydrase, thioredoxin h-type1and a ribonucleoprotein as well as five positive regulators, including genes encoding a serine/threonine-protein kinase, poly(U)-specific endoribonuclease-B, DnaJ homolog subfamily C member2, a GTP-binding protein and NtTRXh. Interestingly, the ribonucleoprotein played a distinct role in the Cf-4/Avr4-mediated and the Xoo-induced HR.5. The role of Agrobacterium to repress Xoo growth and the Xoo-induced HR and its mechanisms were elucidated. Our study demonstrated that four Agrobacterium strains showed diverse roles in inhibiting the Xoo-induced HR. Strains GV3101, EHA105and LBA4404strongly suppress this HR while strain C58C1display no effect on this HR. The mechanism used by the three strains to inhibit the HR was distinct. Strain GV3101showed strong antibiotic role and thus directly inhibited the growth of Xoo employing a mechanism involving hrcU gene from bacterial type three secretion system (T3SS) of Xoo and Ti plasmid, pTiC58, from GV3101. While strains LBA4404and EHA105did not show antibiotic role, rather blocked ROS accumulation thereby repressed the Xoo-induced HR in N. benthamiana.Collectively, in this study, we at the first time unveiled the two layers of mechanisms underlying the expression regulation of the two R gene Cf-4and Cf-9at transcriptional and post transcriptional levels. A miRNA targeting both Cf-4and Cf-9was identified. Moreover, employing cDNA library VIGS screening, we identified a set of novel regulatory genes that were involved in the Xoo-induced nonhost resistance and the Cf-4/Avr4-mediated HR. In addition, we firstly revealed the inhibitive role of Agrobacterium in Xoo itself and its induced HR in plant. These results provide some insights into the mechanisms of host ETI and nonhost resistance.