Preliminary Studies On The Function Of AaSIP2 And AaSNF4 Genes In The Tangerine Pathotype Of Alternaria Alternata

Alternaria brown spot(ABS),caused by Alternaria alternata pathotype tangerine,is one of the major fungal diseases of citrus.It mainly infectes the young leaves,twigs and fruits of the susceptible citrus varieties,which significantly decreases the quality and quantity of tangerine and its hybrid,resulting in heavy losses of the citrus industry.At present,the genome sequencing of Alternaria alternata pathotype tangerine has been completed,which is crucial for further exploration of the pathogenic mechanism of A.alternata.The sucrose non-fermenting 1(SNF1)protein kinase complex is a highly conserved heterotrimeric protein,including a catalytic subunit ? and two regulatory subunits(?,?),which plays an important role in fungal development and virulence.Our previous study revealed that catalytic subunit ? of the A.alternata plays a crucial part in the utilization of alternative carbon sources and virulence.However,the biological functions of the other two subunits have not been reported.In order to understand the functions of other subunits of SNF1 complex in the tangerine pathotype of A.alternata,AaSIP2 and AaSNF4 were identified using the Saccharomyces cerevisiae regulatory subunits(?,?)amino acid sequences as a query to search the complete genome of A.alternata Z7 strain by BLASTP.Based on the targeted-gene knockout strategy and phenotypic analyses,combined with the bioinformatics and real-time PCR analysis,we identified and characterized the AaSIP2 and AaSNF4 of A.alternata.The main results are showed as followed:1.The AaSIP2 contains 1 800 bp ORF,which encodes a protein of 600 amino acids.The AaSNF4 contains 1 086 bp ORF,which encodes a protein of 362 amino acids.Physical and chemical analyses showed that the AaSip2 was a stable hydrophilic protein which the molecular weight was 64.43222 KDa,PI was 6.1,instability index was 53.90 and the grand average hydropathicity(GRAVY)was-0.734.The AaSnf4 was an unstable hydrophilic protein which the molecular weight was 40.7148 KDa,PI was 6.1,instability index was 35.01 and GRAVY was-0.162.The secondary structure analysis showed that the AaSip2 protein was mainly composed of random coils,but there was no ?-turn.The AaSnf4 protein was mainly composed of a-helix and random coil.There was no transmembrane domain and signal peptide in AaSip2 and AaSnf4 protein,which is speculated that these two proteins are neither the membrane protein nor the secretory protein.AaSip2 and AaSnf4 were located in cytoplasm,it is speculated that these two proteins may play a major role in the cytoplasm.Both AaSip2 and AaSnf4 were geneticly closer to the proteins of Pyrenophora tritici-repentis respectively,which showed that they shared higher homology.2.Split-marker strategy was used to construct AaSIP2 and AaSNF4 gene knockout box,and PEG-mediated protoplast transformation was carried out to gain gene knock-out mutants.The mutants were picked out after screening and verification by hygromycin,PCR and southern blot.The efficient homologous recombination mechanism in S.cerevisiae was used to construct the complementation plasmid,and we got the complementary transformants after transformation.The complementary strains CP-Si-4 and CP-Sn-3 were obtained from medium supplemented with antibiotic G418 and examined by PCR.3.Real-time quantitative PCR was used to analyze the expression pattern of AaSIP2 and AaSNF4 under different conditions.AaSIP2 and AaSNF4 were consistently expressed during the process of infecting host by A.alternata Z7 strain,indicating that AaSIP2 and AaSNF4 genes may participate in the host defense response during the process infection.In addition,the expressions of AaSIP2 and AaSNF4 were also different under different carbon source culture conditions.The highest expression was obtained when maltose as the single carbon source,followed by sucrose.On the contrary,AaSIP2 and AaSNF4 showed the lowest expression level in the Na AC medium.It indicated that AaSIP2 and AaSNF4 have important role in the utilization of alternative carbon source in A.alternata.4.AaSIP2 and AaSNF4 are required for sporulation,sugar and partial salt stress response,utilization of alternatuive carbon source and virulence of A.alternata.The deletion of AaSIP2 or AaSNF4 affected the spore morphology,germination and sporulation of A.alternata.The conidia of ?AaSip2 and ?AaSnf4 mutants became longer.Compared with wild type and complementation strains,the amount of conidia and germination decreased signifantly of ?AaSip2 and ?AaSnf4 mutants.Moreover,the mutants increase sensitivity to sugar,Ca Cl2 and KCl stress,and the growth rate was inhibited to different extents in MM medium with different single carbon sources,especially in maltose and sucrose medium.Pathogenicity tests revealed that AaSIP2 and AaSNF4 are essential for fungal virulence.The gene deletion mutants of AaSIP2 or AaSNF4 were reduced in their ability to induce necrotic lesions on detached tangerine leaves.