The Circadian-related Protein XCT Positively Regulates RPW8.1-mediated Immunity In Arabidopsis

Powdery mildew is one of the most common and harmful parasitic fungi disease in plant.The major method of controling powdery mildew disease is through chemical regulation.However,the Pesticide residue and enviromental pollution are becoming increasingly serious,so the most effective and safest way is to find resistant germplasm resources and to breed resistant varieties.Most of the plant resistant?R?genes are race-specific resistance,which are easy to lose their resistance due to the loss or variation of pathogen AVR genes.Therefore,it is urgent to find and explore broad-spectrum resistance genes.The Arabidopsis thaliana powdery mildew resistance locus RESISTANCE TO POWDERY MILDEW8?RPW8?contains two homologous genes RPW8.1 and RPW8.2,which can alone or combined together to resistant Arabidopsis powdery mildew,downy mildew,N.tabacum powdery mildew,Cauliflower mosaic virus and other pathogens,so it is a broad-spectrum resistance gene.Previous study found that RPW8.2 can specifically target to the extrahaustorial membrane and mediated resistance to powdery mildew by a SA-dependent pathway.Although RPW8.1 is localized around the chloroplast in mesophyll cells,it can also resistant to powdery mildew,downy mildew,bacterial,even the rice Pyricularia oryzae and Xanthomonas oryzae.However,the regulation mechanism of RPW8.1 remains unclear.In order to identify the regulator of RPW8.1-mediated broad-spectrum resistance,we performed a forward genetic screen for the homozygous transgenic line R1Y4 which express RPW8.1-YFP from its native promoter using ethyl methanesulfonate?EMS?mutagenesis treatment.A mutant?b3-17?was screened out from numerous mutants which exhibited yellowish freshly emerged leaf,compromised cell death and smaller plant stature in comparison with R1Y4.Then an F2 segregating population was generated by crossing b3-17with Landsberg erecta?Ler?.Finally,by combining map based cloning and genome re-sequencing technologies,we identified a single base G-to-A point mutation at the end of 3^'splice site of 8^th intron in At2g21150.Further sequencing XCT cDNA from b3-17 revealed that this mutation resulted in a splicing shift of eight nucleotides downstream of the original splice junction,generating a mRNA encoding a truncated XCT lacking the C-terminal 79amino acid residues that covers one-third of the conserved X-chromosome Associated Protein5?XAP5?domain.By introducing the wild type XCT gene into b3-17,the positive lines were all restored to the phenotypes of R1Y4.So far,we identified the mutant-related gene XCT,and renamed b3-17 as R1Y4/xct-5.We also made the constructs expressing an artificial microRNA or overexpressing XCT and introduced them into R1Y4 and Col-gl,respectively.Subsequently,We investigated the disease resistance of this gene alone and its effects on RPW8.1 mediated disease resistance,respectively.The main results are as follows:1)Mutantion in XCT compromised RPW8.1-mediated immune response.First,the sporulation on b3-17 was significantly higher than that on R1Y4 at 10 days post inoculation with powdery mildew,indicate the impairment of RPW8.1-mediated resistance against powdery mildew in the b3-17 mutant.Then we examined some defensive responses including the distribution of cell death and production of H₂O₂ upon powdery mildew infection,callose deposition and expression of defense-related genes induced by flg22.All the responses were compromised in mutant.Additionally,proliferation of both P.syringae DC3000 and P.syringae DC3000?hrcC^-?in b3-17 was significantly higher than that in R1Y4.Together these data indicate that XCT is required for RPW8.1-mediated defensive responses and resistance to different pathogens.2)When down-expression of XCT in R1Y4,the cell death lesions were obviously less severe in R1Y4/amiRXCT lines than those of R1Y4,and the resistance against powdery mildew was also weakened in R1Y4/amiRXCT lines.The cell death and H₂O₂ induced by powdery mildew,callose deposition and expression of FRK1 induced by flg22 were all reduced in R1Y4/amiRXCT lines.These results suggest that lower expression of XCT in R1Y4compromised RPW8.1-mediated defensive responses,implying XCT acts as a positive regulator for RPW8.1-mediated resistance.3)The overexpression lines displayed enhanced cell death than R1Y4 in the absence of pathogen,and displayed enhanced resistance to powdery mildew and P.syringae.The related defense responses such as cell death,H₂O₂ accumulation,callose deposition,defense gene expression were all increased in overexpression lines.These results suggest that over expression of XCT enhanced RPW8.1 mediated resistance and emphasize the positive role of XCT in RPW8.1-mediated immunity.4)Both the mutant,interference lines and overexpression lines of XCT in Col-gl background displayed similar disease phenotypes of powdery mildew and P.syringae to wild type,implying that XCT alone may not be involved directly in defense.5)We examined the mRNA level and protein level of RPW8.1 by qRT-PCR and western blot,respectively.The results indicate that XCT positively regulate RPW8.1 expression at both transcriptional level and protein accumulation.Meanwhile,RPW8.1 can also up-regulate the expression of XCT,indicating that both are somehow mutually up-regulated at the transcriptional level.6)The expression of ethylene-signaling transcription factors ERF6,ERF016 and ORA59 was inhibited in R1Y4 and R1Y4/OEXCT,but up-regulated in R1Y4/xct-5,respectively.This suggests that ethylene signaling may be involved in XCT and RPW8.1 mediated disease resistance processes.Taken together,our results suggest that XCT positively regulate RPW8.1-mediated cell death and disease resistance through suppressing ET-signaling pathway,and provide insight into the regulatory mechanism of RPW8.1-meidated immunity.