Functional Analyses Of Two Arabidopsis WRKY Genes And Nicotiana Benthamiana Glycolate Oxidase Genes In Disease Resistance

Reactive oxygen species (ROS) is an essential regulator of plant disease resistance. Two pathways are currently known to generate ROS, generation of superoxide anion by NADPH oxidase and produce of H2O2 by glycolate oxidase (GOX). Recently, it is reported that WRKY transcription factor may regulate ROS during effector-triggered immunity (ETI) and PAMP-triggered immunity (PTI). A number of WRKYs play roles in plant disease resistance but exhibit diversified effects. To date, function of Arabidopsis WRKY6 (AtWRKY6) in plant disease resistance remains unclear and role of AtWRKY8 in plant resistance is not well understood. Function of these two WRKYs in ROS is not clear. In addition, role of GOX in plant resistance depends on the pathogen. In this thesis study, functions and mechanisms of AtWRKY6, AtWRKY8 and Nicotiana benthamiana GOX genes in ROS accumulation and resistance to Sclerotinia sclerotiorum (Ss) and Xanthomonas oryzae pv. oryzae (Xoo) were analyzed. The obtained main results are as follows:(1) Function of AtWRKY6 and AtWRKY8 genes in resistance to Ss and Xoo were explored. Inoculation results using knockout mutant and overexpression transgenic plants demonstrated that both AtWRKY6 and AtWRKY8 positively regulated resistance to Ss, however, they played different roles in nonhost resistance to Xoo with a positive role for AtWRKY6 but a negative role forAtWRKY8.(2) The mechanisms underlying AtWRKY6 and AtWRKY8 mediated defense were elucidated. DAB staining analysis of knockout mutant and overexpression transgenic plants indicated that AtWRKY8 positively while AtWRKY6 negatively regulated Xooinduced ROS accumulation. Gene expression analysis by qRT-PCR showed that AtWRKY6 and A1WRKY8 modulated expression of RbohD, RbohF, GOX1 and HAOX1 constitutively and at early phase of Xoo infection (6 hpi) thereby regulating ROS accumulation. Moreover, detection of ROS in knockout mutant and overexpression transgenic plants revealed that AtWRKY6 positively regulated bacterial PAMP (flg22) elicited ROS, while negatively modulated fungal PAMP (chitin) and Arabidopsis DAMP (AtPep1) elicited ROS. However, AtWRKY8 negatively regulated ROS elicited by all above PAMPs and DAMP. This result indicates that AtWRKY6 and AtWRKY8 are invovled in PTI and DTI. Aniline blue staining assay showed that AtWRKY6 positively while AtWRKY8 negatively altered callose deposition in response to Ss infection. Differential transcriptome profiling of overexpression (35S:WRKY6-9) and knockout mutant (wrky6) demonstrated that AtWRKY6 functioned in constitutive resistance through promoting SA pathway involving SA transport and signaling genes SARD1, EDS1, EDS5, NIMIN-1, NIMIN-2, and NPR2-5. In response to Ss infection, AtWRKY6 downregulated a variety of resistance negative regulators such as MLOs, EDR2, BON3, BAPs and NUDT7, thereby promoting resistance to Ss, meanwhile activated Rapid alkalinization factors (RALFs) and GLPs to overcome the great amount of oxalic acid secreted by Ss, and downregulated the FLS2-degrading factors PUB 12/24, chitin-triggered PTI key components LYM2, CERK1, BAK1 and BIK1, and DTI elicitor genes PEP1-3 and their receptor PEPR2, to positive regulate flg22-triggered PTI while negatively modulate chitin-triggered PTI and AtPeps-triggered DTI.(3) GOX gene family was identified in N. benthamiana genome employing bio informatics approaches, and function of three NbGOX genes NbHAOX8, NbGOX1 and NbGOX4 in resistance to Ss and Xoo were investigated using virus induced gene silencing (VIGS). Sixteen NbGOX genes were identified. Sequence analyses indicated that they all possessed an alpha_hydroxyacid_oxid_FMN domain with an extra sequence of 43-45 amino acids after the second putative catalytic site compared to the domain deposited in NCBI-CDD database. VIGS functional analyses demonstrated that NbGOX1 and NbHAOX8 negatively regulated the nonhost resistance to Xoo, not obviously affected the resistance to Ss, while NbGOX4 positively regulated both types of resistance.(4) The mechanisms underlying NbGOXs mediated defense were analyzed. DAB staining results showed that NbHAOX8- and NbGQX1-silenced plants accumulated higher level of ROS, while NbGOX4-silenced plants accumulated lower level of ROS, than eGFP nonsilenced control plants, indicating that NbHAOX8 and NbGOXl negatively while NbGOX4 positively regulated Xoo-induced ROS accumulation. Quantitative detection assays demonstrated that all these three genes negatively regulated flg22-elicited ROS. Expression analysis showed that these genes did not significantly change the expression of RbohD and RbohF, suggesting that ROS regulation by these NbGOXs and Rbohs was independent. Furthermore, silencing of NbGOX4 significantly enhanced expression of NbHAOX8 and NbGOX1, implying that NbGOX4 might function via NbHAOX8 and NbGOXl. In addition, NbGOX1 might use SA pathway, NbGOX4 might employ JA pathway and WRKY45 and WRKY62-mediated pathways, while NbHAOX8 might exploit yet unknown pathways, to regulate plant disease resistance.