| Citrus is one of the world’s important fruit crops.Citrus Alternaria brown spot(ABS),caused by the tangerine pathotype of Alternaria alternata,is a highly destructive disease of citrus worldwide.It affects many tangerines and their hybrids,causing defoliation,twig dieback,and young fruit-dropping,resulting in reduced yield and fruit quality.To date,the most effective control measure of citrus ABS is still chemical control,but chemical control is usually limited during a disease outbreak.Indepth investigations of biological properties and underlying mechanisms of pathogenicity of A.alternata may provide more theoretical basis for the control of ABS and other diseases.Stu A is a member of the fungal-specific APSES transcription factor family,although the roles of Stu A have been elucidated from several fungi,little is known about the downstream target genes of Stu A and related regulatory mechanisms.In this study,we explored the functions of A.alternata AaStuA in ACT biosynthesis and pathogenicity using biochemical,plant pathology and bioinformatics approaches.The study results show that:We identified Stu A homolog in the tangerine pathotype of A.alternata,designating as AaStuA.Deletion of AaStuA resulted in severe defects in growth,conidiation,ACT toxin biosynthesis and pathogenicity.AaStuA has five predicted protein kinase A(PKA)phosphorylation sites,site-directed mutagenesis showed that the conserved predicted T110 site is not important for the nuclear localization of AaStuA,but is important for its function in growth,conidiation.Nevertheless,the ΔAaStuA-CT110 A transformant was similar to the wild-type and complementation transformant with regard to ACT production.The Aa Som1 protein which functions downstream of the c AMP dependent protein kinase A(c AMP-PKA)could interact with AaStuA in the nucleus.Deletion of Aa Som1 resulted in lost of ACT biosynthesis and pathogenicity completely.Deletion of AaStuA in the ΔAa Som1 background(ΔAa Som1-AaStuA)resulted in similar phenotypes as ΔAa Som1.The expression of AaStuA was significantly downregulated in ΔAa Som1,indicating that Aa Som1 acts upstream of AaStuA in A.alternata.Furthermore,we found that there is more than one copy of thep H‐responsive transcription factor-encoding gene Aa Pac C.Silencing of Aa Pac C resulted in severe defects in ACT biosynthetic genes expression,ACT toxin biosynthesis,and pathogenicity.Yeast two-hybrid and GST-Pull down assays confirmed that AaStuA could interact with Aa Pac C in the nucleus.AaStuA regulated the transcriptional expression of Aa Pac C during the ACT toxin-inducing period.EMSA assays revealed that Aa Pac C directly binds to the promoter region of ACTT6 and ACTTR to regulate their transcriptional expression directly.Both the enoyl coenzyme A hydrolase-encoding gene ACTT6 and the Zn2Cys6 transcription factor-encoding gene ACTTR from the ACT toxin biosynthesis gene cluster are essential for ACT biosynthesis.Taken together,this study uncovered that AaStuA regulates ACT biosynthesis of A.alternata by a Aa Som1→AaStuA→Aa Pac C→ACTT regulatory pathway.Deletion of AaStuA resulted in increasing sensitivity to oxidants and decreasing ROS detoxification.AaStuA affected the peroxisomal structural integrity by regulating the transcriptional expression of peroxisome formation-related genes,but had no effect on the peroxisomal localization of proteins containing peroxisome-targeting signals.In addition,yeast two-hybrid assays showed that AaStuA could interact with the peroxisome-regulated transcription factor Aa Msn2.Our results indicated that AaStuA interacted with Aa Msn2 to affect peroxisomes formation and thus the ability to resist oxidative stress and ROS detoxification.Deletion of AaStuA resulted in the activation of autophagy.To further investigate the relationship between cellular autophagy and ACT toxin biosynthesis,we deleted 19 autophagy-related genes of A.alternata.Functional analysis revealed that 12 Aa ATG genes were involved in pathogenicity,but only 5 Aa ATG genes were involved in ACT toxin biosynthesis.The relationship between autophagy and ACT biosynthesis remains to be investigated.RNA-seq comparison of transcripts from ΔAaStuA and the wild-type Z7 strain revealed that AaStuA was involved in the transcriptional regulation of genes related to ribosome synthesis,amino acid synthesis and catabolism,amylose metabolism,nitrogen metabolism,secondary metabolite synthesis,and antioxidant pathway.In summary,our results show that AaStuA is involved in the regulation of ACT biosynthesis by the Som1→Stu A→Pac C→ACTT pathway.The transcription factor AaStuA may interact with Aa Msn2,and affect the ability to resist oxidative stress and ROS detoxification.These works provide a new understanding for the mechanism of ACT biosynthesis,and provide a new theoretical basis for further investigation of the pathogenesis of A.alternata. |
