The Establishment Of The Arabidopsis–Colletotrichum Gloeosporioides Pathosystem And The Preliminary Functional Study On The SgPALs

Stylosanthes is the largest and most widely used leguminous forage as both feed and cover crop in tropical regions.Anthracnose is the most important disease that limits the yield Stylosanthes.At present,basic research on Stylosanthes anthracnose focuses more on the isolation and identification of pathogenic genes related to the pathogen.However,the research on the pathogenic mechanism of Stylosanthes anthracnose and the molecular mechanism of Stylosanthes anthracnose is very limited.Many basic issues have not yet been clarified.In order to speed up the study of the molecular mechanism of anthracnose under the restrictive conditions such as the immature stylus transgenic system.In this paper,an interaction system between Arabidopsis thaliana and Colletotrichum gloeosporioides was constructed.At the same time,this system was used to preliminarily explore the mechanism of resistance to anthracnose and the function of the phenylalanine ammonia lyase gene SgPALs in Stylosanthes.The main work and results are as follows:（1）The Arabidopsis–Colletotrichum gloeosporioides pathosystem was established.First,27 strains that can infect Arabidopsis were separated from the Stylosanthes.According to the percentage of lesion area（lesion area/leaf area）,they were divided into three categories:strong,medium and weak.The pathogenicity of 13 strains was consistent in Stylosanthes and Arabidopsis.Sequence analysis of ITS,ACT,CHS and GADPH genes of co-infected strains and the morphological characteristics of representative isolates showed that the pathogen was Colletotrichum gloeosporioides sensu lato（s.l.）.Finally,the symptom and infection process of strong pathogen（DZ-19,HK-04）and weak pathogen（CJ-04）in different Arabidopsis ecotypes and developmental stages were compared.The results showed that the disease caused by strong pathogens was more serious than weak pathogens,and the infection cycle was faster.（2）The mechanism of plant resistance to Stylosanthes anthracnose has been preliminarily explored.Based on the interaction system established in this paper,the genetic analysis of Arabidopsis defense response mutants and the expression profile analysis of defense marker genes were carried out.The results show that mitogen-activated protein kinase（MAPK）,Ca²⁺dependent protein kinase（CDPK）,salicylic acid（SA）,ethylene（ET）and jasmonic acid（JA）pathways are all involved in the process of plant resistance to anthracnose.（3）The functions on resistant to disease of the Stylosanthes phenylalanine ammonia lyase genes SgPALs was preliminarily explored.By constructing the transgenic Arabidopsis thaliana overexpressing SgPALs and using the interaction system established in this article,the SgPALs transgenic lines were more resistant than the wild type.In terms of disease-resistant phenotype,the lesions of the over-expression lines were significantly smaller than those of the wild type.At the physiological level,the accumulation of secondary metabolites,including total phenols and flavonoids,in overexpression lines increased.At the molecular level,the expression of disease resistance-related genes in the MAPK,CDPK,ET,and JA signaling pathways of the overexpressing lines increased.In conclusion,an interaction system between Arabidopsis thaliana and Colletotrichum gloeosporioides was constructed,and it was used to explore the resistance mechanism of Arabidopsis to Colletotrichum by analyzing the expression patterns of marker genes related to disease resistance and defense response of defense mutants.This interaction system was used to preliminarily analyze the mechanism that SgPALs may enhance plant resistance to anthracnose by increasing the accumulation of secondary metabolites,total phenols and flavonoids.The disease resistance process also involves the regulation of MAPK,CDPK,ET and JA pathways.These results will lay the foundation for exploring the mechanism of plant disease resistance and breeding of resistant varieties.