Exploration Of Citrus Canker Resistance Genes And Improvement Of Gene Editing System

Citrus canker,caused by(Xanthomonas citri subsp.citri,Xcc)bacteria,seriously threatens the development of citrus industry.Almost all citrus cultivars are susceptible to canker disease.Citrus has a broad spectrum of resources ranging from primitive,wild,and cultivated status.Currently,little is known about the resistance or susceptibility of primitive or wild citrus to canker disease.To explore the canker resistant germplasm,we expanded the research scope to investigate wild citrus,and found that Atalantia is highly resistant to citrus canker.We systematically studied the molecular mechanism of canker resistance of Atalantia.Meanwhile,we explored defense-related genes in Atalantia by comparative transcriptomics and investigated candidate genes function.In addition,the process of traditional breeding in citrus often faced with a lot of problems,such as abortive ovule,polyembryony,and prolonged juvenility.The development of CRISPR/Cas9 technology accelerating the process of citrus molecular breeding,it has been applied in citrus canker-resistance breeding.However,citrus gene editing system often faced with several challenges,such as the lower transformation and editing efficiency.Researches on how to optimize the citrus gene editing system is rare.In this study,we optimized the application of PTG/Cas9(polycistronic t RNA-sg RNA/Cas9)system in citrus gene editing.The main research results are as following:1.Canker disease evaluation of wild citrus germplasm.To explore the canker resistant germplasms,we investigated 10 wild and cultivated citrus in tribe Citrinae belonging to the subfamily Aurantioideae.Three wild citrus varieties including wild mandarin,wild pummelo,and citron showed different degrees of susceptibility to citrus canker,wild resource purple pummelo was more sensitive to citrus canker.However,remarkable resistance was observed in Atalantia buxifolia,a distant wild relative of citrus.2.Identification of differentially expressed genes of Atalantia in response to canker infection.By analyzing the transcriptome data of Atalantia and sweet orange post-inoculation with Xcc,we found that the up-regulated genes in Atalantia were enriched in pathways such as defense response and biotic stress response,while those in sweet orange were enriched in polysaccharide metabolic and catabolic pathways at 48 h after the inoculation.3.Elucidation the regulatory mechanism for the resistance of Atalantia to Xcc.Three of amino acids encoded by transcription factorTFIIA? in Atalantia(AbTFIIA?)exhibited difference from those in sweet orange(CsTFIIA?)which could stabilize the interaction between effector Pth A4 and effector binding element(EBE)of LOB1 promoter.Luciferase complementation and GST pull down assay showed that the mutation of AbTFIIA? did not change its interaction with transcription factor binding motifs(TFBs).However,the functional complementation assay indicated that AbTFIIA? could hardly support the LOB1 expression induced by the Pth A4.In addition,sequence alignment of AbLOB1 and Cs LOB1 revealed that two nucleotide differences between the two promoters EBE.Further promoter activity assay showed that the activity of AbLOB1 promoter was significantly lower than that of Cs LOB1 under the induction of Pth A4.Our results demonstrate that natural variations of AbTFIIA? and effector binding element(EBE)in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia.Therefore,we speculated that Atalantia could enhance its resistance to citrus canker by reducing the expression of the susceptible gene LOB1 and activating resistant genes expression.The natural mutations of AbTFIIA? gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease.4.Characterization of the defense-related genes from Atalantia.We analyzed the transcriptional differentially expressed genes in Atalantia and sweet orange leaves after inoculation with Xcc,the result showed that the expression of COMT was significantly up-regulated in Atalantia,while the expression was not significantly changed in the susceptibility sweet orange.AbCOMT-overexpressed citrus increased the content of melatonin and resistance to citrus canker to some extent.5.Optimization of highly efficient gene-editing system in sweet orange.We obtained 73 CsPDS gene edited seedlings and realized multiple target sites editing by stable transformation of sweet orange epicotyls with different PTG/Cas9 vectors driven by YAO promoter.By optimizing the genetic transformation system of sweet orange,the transformation efficiency of Anliu sweet orange was greatly improved.The mutation efficiency of CsPDS gene was significantly improved by heat stress treatment in this study.Our results provided a useful tool for the multiplex-gene editing of defense-related genes for the next step.