Functional Analysis Of Arabidopsis WRKY And AGO1Genes In Regulation Of Resistance To Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum, one of the most destructive plant pathogenic fungi, causes white mould disease, resulting in huge yield loss every year, and thus is a serious problem requiring to be solved urgently in agricultural production. So far, the molecular mechanism underlying plant resistance to S. sclerotiorum is still largely unknown. In this study, to provide some insights into this resistance mechanism, and obtain some potentially useful gene resources for molecular breeding for resistance to S. sclerotiorum, using model plant Arabidopisis thaliana, function of the WRKY gene family and the AGO1gene in regulation of resistance to S. sclerotiorum is analyzed. The main results are as follows:(1) Subcellular localization of all Arabidopsis AGO members were predicted using bioinformatics methods, and that of one member, AtAGO2, was experimentally identified. All ten AtAGOs did not carry signal peptide, indicating that they were not secretory proteins. AtAGOl, AtAGO2, AtAGO3, AtAGO4and AtAGO9contained nuclear localization signals, although differing in sequence, demonstrating that they might be localized in cell nucleus. The full-length cDNA sequence of AtAGO2was cloned and the expression vector pCHF3-eGFP::AtAGO2in which AtAGO2was fused with eGFP reporter gene was constructed to identify the subcellular localization of AtAGO2. AtAGO2was transiently expressed by Agrobacterium-mediated method in leaves of Nicotiana benthamiana. Results of detection of green fluorescence signal by confocal laser-scanning microscope showed that AtAGO2was localized in cytoplasm, plasma membrane and nucleus. Jasmonic acid, but not the resistance inducer BTH, rendered AtAGO2gather as bright "granules" in and around plasma membrane.(2) Arabidopsis AGO1overexpression transgenic plants (AtAGOl-OE) were generated. Using these AtAGOl-OE plants together with agol mutants, role of the AGO] gene in regulation of resistance to S. sclerotiorum was clearly revealed. The overexpression construct35S::AtAG01was constructed and introduced into Col-0wild-type plants following floral dip transformation protocol.35S::AtAGOJ transgenic lines with single copy of the tansgene were obtained. After selection of three generations, homozygous lines were isolated. qRT-PCR analysis showed that AGO] gene expression level differed in lines, some were over5times as high as in Col-0, some were just1or2times while some others were lowered to only0.15times as in wild-type plants.Function of the AGO1gene in regulation of resistance to S. sclerotiorum was analyzed using agol mutants and AtAGO1-OE plants. Compared with the wild-type plants, disease symptoms of agol-27were more severe while those of AtAGO1-OE lines were weaker. Results of qRT-PCR detection for PR gene expression showed that in AtAGO1-OE plants, constitutive expression of PR1, PAD4and RD22were higher than in Col-0. After Ss inoculation, expression of PR5was down-regulatd in early time point while the other fives (PR1, PAD4, GST, PDF1.2and RD22) were all up-regulated in wild-type plants. However, in AtAGO1-OE plants, expression of all six genes were up-regulated, whereas in agol mutants, expression of PAD4and PR5were down-regulated in early and later time-point respectively, PR1and RD22up-regulated in later time-point, while G5T and PDF1.2up-regulated constantly after inoculation, but in a magnitude less than in AtAGOl-OE plants. These results revealed that AtAGO1positively regulated resistance against S. sclerotiorum by activating multiple signaling pathways in a variety of ways.(3) By using mutants, role of Arabidopsis WRKY family in regulation of resistance to S. sclerotiorum was analyzed. Expression of AtWRKY6, AtWRKY8and AtWRKY11were highly induced in response to Ss infection. Compared with wild-type plants, disease symptoms of wrky6and wrky8mutants were more severe while those of wrky69and wrky70mutants were weaker. Trypan Blue staining analysis showed that Ss mycelia grew faster and thus with higher biomass in wrky6and wrky8plants, while slower and with less biomass in wrky69and wrky70mutants in comparisom with that in wild-type plants. DAB staining data demonstrated that H2O2accumulated much more in wrky6and wrky8mutants while less in wrky69and wrky70mutants compared with that in wild-type plants. Defense gene expression results showed that, compared with wild-type plants, constitutive expression of NPR1gene was reduced while that of PR5increased in wrky6and wrky8mutants, however, expression of these two genes and PAD4were all up-regulated in wrky69and wrky70mutants, After Ss inoculation, expression of PR1, PR5, PAD4, NPR1and PDF1.2were all up-regulated obviously in wrky70. Similarly expression of PR5, PAD4, NPR1and PDF1.2in wrky69were also strongly increased although PR] only weakly up-regulated. However, expression of these genes in the wrky6and wrky8mutants were either down-regulated (PR5and PAD4) or up-regulated only in a magnitude less than in Col-0and much less than in wrky69and wrky70(PR1, NPR1and PDF1.2). These results revealed that AtWRKY6and AtWRKY8were positive regulators, while AtWRKY69and AtWRKY70negative ones of resistance to S. sclerotiorum that functioned by activating multiple signaling pathways.