Identification And Functional Analyses Of Effector Proteins Of Alternaria Alternata

Citrus have the large trade volume in the world and second only to banana,but it is often threatened by various pests and diseases in the process of production.Among them,the Alternaria brown spot(ABS)caused by Alternaria alternata pathotype tangerine is a kind of fungal disease which seriously endangers the young shoots,leaves and fruits of citrus,causing serious problems for the production of the susceptible citrus varieties.Because citrus brown spot disease is a new disease in China,the outbreak disaster problem has only begun to appear prominent.We lack a systematic understanding of the pathogenesis of A.alternata pathotype tangerine.The effector protein is a molecular substance secreted by pathogen during infection of the host.The effector plays a key role in the long-term interaction between plants and pathogen.The research systems of bacterial and oomycete effector proteins are relatively mature,and some research progress has been made.The study of effector proteins in pathogenic fungi is weak.There are less reports on the effector proteins of citrus brown spot pathogens.The research on the effector proteins of A.alternata will reveal the molecular interactions between A.alternata and citrus,which will help us understand the causes of citrus disease and disease resistance at the genetic level,and even provide more opportunities for the cultivation of citrus disease resistant varieties and control of citrus brown spot.A comprehensive theoretical strategy will also provide reference for the study of other citrus pathogenic fungi.Therefore,based on the genome sequencing of A.alternata completed by previous researchers,we screened and identified effector genes through bioinformatics softwaresystems and tobacco transient expression system,and conducted preliminary studies on their functions.The main result are as follows:1.A total of 256 candidate effector genes were obtained through the genome prediction of A.alternata,and transient expression screening of 3 effector proteins Aa SP4,Aa SP16,Aa SP28 in tobacco cells induced induction of allergic necrosis in tobacco leaf tissue,Aa SP1,Aa SP3,Aa SP5,Aa SP6,Aa SP7,Aa SP13,Aa SP17,Aa SP20,Aa SP22,Aa SP27 and Aa SP29 11 effector proteins can inhibit PCD caused by Bax.The 14 effector proteins had 127-308 aa,molecular weights in 12.54-34.01 k Da,and p I ranged from 3.79 to 6.73.Except for Aa SP1,the other effector proteins had a signal peptide at the N-terminus.The cleavage sites were between 16 and 21 between amino acids;Subcellular localization predictions revealed that Aa SP28 was in mitochondria,Aa SP1 was in other unknown structures,and the remaining 12 effectors were secreted extracellularly;Aa SP13 was annotated with a Cutinase domain protein,Aa SP16 was annotated as Xylanase and contained GH11 in the domain,Aa SP20 was annotated as an allergic reaction-inducing protein,CFEM domain was present in Aa SP29,and the remaining 10 effector proteins are hypothetical proteins.2.The effector genes AaSP4,AaSP16 and AaSP28 had homologous genes in other non-Alternaria genome.The genes most closely matched with them were from A.alternata,indicating that they had a conservative biochemical function.In addition,the polymorphisms of these 3 effector genes in the different physiological races of citrus brassica were not high,indicating that they were conservative during the evolution of citrus brassica.3.The expression patterns of AaSP4,AaSP16 and AaSP28 were different in the host infected by A.alternata.Aa SP16 showed stable expression during the infection process;Aa SP4 was up-regulated after 48 hours;Aa SP28 was down-regulated,presumably Aa SP16,Aa SP4,Aa SP28 may participate in the early,middle and late stages of the interaction between A.alternata and host,respectively,and regulate the host defense response.4.AaSP4,AaSP16 and AaSP28 did not find a common motif among the three effector proteins.Sequence deletion mutations were performed on them and found that the N-terminal signal peptide and the C-terminal 139-206 amino acid sequence of Aa SP4 were necessary to induce the cells death.;the N-terminal signal peptide and the C-terminal GH11 domain of Aa SP16 played an important role in inducing necrosis of tobacco tissues,and the 19 amino acids at the C-terminus were essential for Aa SP28 to induce the cells death.5.The subcellular localization of AaSP4,AaSP16 and AaSP28 in onion epidermal cells showed that they were located on the intercellular substance.It was speculated that Aa SP4,Aa SP16 and Aa SP28 may play a role in cell membrane.