Identification And Functional Analysis Of The Effector Protein Induced By Alternaria Alternate

Alternaria brown spot(ABS),caused by Alternaria alternata pathotype tangerine,is a major fungal disease that seriously damages citrus.The disease spreads very fast and occurs in a wide range,which has a serious impact on the production of susceptible citrus varieties.However,Alternaria brown spot is a new disease in China,so there is still a lack of systematic and in-depth understanding of the pathogenic mechanism of the pathogen,leading to the current control of Alternaria brown spot has been in a passive state.Effector protein is a small molecule secreted by pathogen in the process of infecting host,which can enhance its own virulence.It plays a key role in the long process of interaction between plant and pathogen.Up to now,the research system of effector protein in bacteria and oomycetes has tended to mature,but in pathogenic fungi,the research progress of effector protein has been very slow,and there are few reports about effector protein in Citrus brown spot.The research on the effect protein of Citrus brown spot pathogen can reveal the molecular interaction between the pathogen and the citrus,so that we can better understand the cause of the gene level in the disease susceptibility and disease resistance of the citrus,at the same time,this research also provides a strong theoretical guidance for the prevention and control of Citrus brown spot and the cultivation of Citrus resistant varieties,which will play a role in the research of other citrus pathogenic fungi It can be used for reference.In this paper,based on the sequencing of the whole genome of chloasma and the previous screening of some effector proteins,we systematically screened the remaining candidate effector genes,analyzed the toxic and non-toxic functions of effector genes,analyzed the effector gene functions of inducing host cell death and inhibiting Bax induced PCD,and analyzed its mechanism of action and interaction with citrus Possible role in the work.The main results are as follows:1.The results showed that the overexpression of 18 effector proteins(Aa SP4 、Aa SP16、Aa SP28、Aa SP43、Aa SP52、Aa SP93、Aa SP114、Aa SP152、Aa SP163、Aa SP55、Aa SP97、Aa SP138、Aa SP38、Aa SP131、Aa SP136、Aa SP71、Aa SP128、Aa SP151)in tobacco cells induced hypersensitivity In the early stage of our laboratory,some studies have been done on the effect of Aa SP4、Aa SP16、Aa SP28.Four effector proteins of Aa SP6、Aa SP13、Aa SP162 and Aa SP64 can inhibit PCD induced by Bax.2.Through bioinformatics analysis,it was found that the eighteen effector genes that can induce the death of the host cell of Citrus brown spot have no homologous genes in other non-Alternaria genomes,indicating that these effector genes are conservative in biochemical functions.Through intra-species sequencing,it was found that the 18 effector genes were not polymorphic in different physiological races of Citrus brown spot,indicating that the effect protein of the brown spot pathogen has a relatively high Conservative.3.Bioinformatics analysis revealed that there was no common motif among the 18 necrotic effect protein genes except for the signal peptide,but Aa SP67,Aa SP114,Aa SP136,Aa SP152,Aa SP162,Aa SP151,Aa SP128,Aa SP64,Aa SP93,Aa SP16,Aa SP138,Aa SP28 Aa SP71,Aa SP43,Aa SP55,Aa SP52,Aa SP97 share a common motif1;Aa SP114,Aa SP152,Aa SP162,Aa SP16,Aa SP71,Aa SP163 share a common motif2;Aa SP114,Aa SP151,Aa SP28,Aa SP163 share a common motif3,Aa SP114,Aa SP14,Aa SP67,aa114 There is a common motif4 among Aa SP136,Aa SP128 and Aa SP64.4.Real-time fluorescence quantitative PCR showed that all 18 effector genes could induce expression during the infection of red citrus by the brown spot pathogen of citrus,and the peak value was 12 h after inoculation.Among them,the expression levels of Aa SP16,Aa SP38,and Aa SP71 were significantly up-regulated after inoculation.At 6h,the relative expression levels of Aa SP16 and Aa SP71 reached nearly 20 times,and these3 genes showed continuous induced expression during the infection process;Aa SP128 was the only gene The expression can be monitored at the 12 th hour;the gene Aa SP97 exhibits a down-regulated expression pattern during the citrus brown spot infection of citrus;the gene Aa SP152 has a relatively small difference in expression throughout the infection process and has been stable throughout the process.These results indicate that the effect protein genes of Citrus brown leaf spot show different expression patterns,and it is predicted that these 18 secreted protein genes may be involved in the identification of the early,middle and late stages of the interaction between the brown spot spot fungus and the host and the regulation process of the host defense response.5.Using the Split-marker method,9 key effector proteins Aa SP52,Aa SP16,Aa SP163,Aa SP6,Aa SP13,Aa SP138,Aa SP128,Aa SP43,Aa SP71 gene knockout cassettes were constructed,and deletion mutants of each gene were obtained by transformation.Observation of colony morphology and mycelial growth rate revealed that some mutant genes with deletion of genes on PDA medium had pigment mutations and some had melanin deposition.The deletion mutants of Aa SP52,Aa SP128,Aa SP71 genes partly showed a decrease in growth rate,and the growth rate of partially deleted mutants was almost the same as that of wild-type strains,and some deletion mutants even showed an increase in growth rate;Aa SP163,Aa SP138,Aa SP43,Aa SP16 deletion mutants all showed a decrease in growth rate,indicating that the deletion of these genes has hindered the growth of the brown spot pathogen.Through environmental stress experiments on mutants,it was found that the deletion mutants have different adaptability to adversity stress.Compared with the wild control,the growth rate of all deletion mutants on the normal medium is slower,and the deletion mutants of the Aa SP128 gene are all resistant to stress.Sensitive;Aa SP138 and Aa SP16 gene deletion mutants are not sensitive to the stresses of Congo red and sorbitol,but are more sensitive to other stresses.The pathogenicity test results showed that except for the Aa SP6 and Aa SP13 genes that inhibited BAX-induced PCD reaction,the pathogenicity of all deletion mutants of effector protein genes that could cause tobacco leaf necrosis was significantly reduced.The above results indicate that these effector protein genes are involved in regulation Pathogenicity of citrus brown spot pathogen.6.The yeast two-hybrid experiment was carried out on the four effector genes,and the interaction protein of the target protein was selected by the yeast two-hybrid system.Finally,after sequencing analysis,the bait Aa SP16 screened 35 positive clones and bait Aa SP52 that interacted with the bait protein 17 positive clones interacting with bait protein were screened,19 positive clones interacting with bait protein were screened by bait Aa SP128,and 28 positive clones interacting with bait protein were screened by bait Aa SP163.7.Bioinformatics analysis,intra-species and inter-species polymorphism analysis,gene expression analysis,gene knockout of two effector protein genes Aa SP6 and Aa SP13,which can inhibit the PCD response induced by BAX,The sequences within the species are relatively conservative.Both of these two effector genes can be induced to express during the infection of Citrus brown spot by red orange,and their peak values are obviously up-regulated at 36 h and 48 h after inoculation.By comparing the deleted mutant strain with the wild-type strain,it was found that the deletion of these two genes had little effect on the pathogenicity,and may play a role in other aspects.