Preliminary Studies On The Function Of SNF1 And FTR1 Genes In The Tangerine Pathotype Of Alternaria Alternata

Citrus Alternaria brown spot(ABS),caused by the tangerine pathotype Alternaria alternata,is a major problem on some tangerines,hybrids of tangerine and grapefruit or tangerine and orange,as well as grapefruit.It causes defoliation,twig dieback,young fruit-dropping,and dramatically market value reduction of infected fruits,especially on susceptible citrus varieties.The tangerine pathotype of Alternaria alternata produces the host-selective ACT toxin,which has been demonstrated to be essential for fungal pathogenesis.Studies have demonstrated that the ability to mitigate the toxicity of ROS is also required for A.alternata pathogenicity/virulence in citrus.However,research on the biosynthetic mechanism of ACT as well as the molecular mechanism leading to ROS detoxification is still not systematic and in-depth.At the same time,the correlational advance in basic metabolism is much not sufficient up to now.Sucrose non fermenting protein kinase SNF1 plays important roles in fungal development and activation of catabolite-repressed genes.Additionally,it also plays important function in various processes related to development,including fungal meiosis,sporulation,filamentous growth,and biofilm formation.For a number of filamentous pathogenic fungi,the SNF1 homologous genes have been shown to be critical for fungal development and virulence.Transcriptome sequencing data showed that the expression of Aa SNF1 was induced by H2O2.The requirement of siderophore for iron acquisition and the involvement of iron tolerance to ROS as well as the iron-dependent antioxidant activities suggest a role for iron in A.alternata pathogenesis.At the low-as compared with the high-iron condition,the defect in ROS sensitivity is due to lack of the ability to acquire iron.Fe transporter permease FTR1 is the permease component of the Fet3p–Ftr1p high affinity iron-uptake complex,in the plasma membrane of Saccharomyces cerevisiae,which transports the Fe3+ produced by the Fet3 p ferroxidase into the cell.To better understand the molecular mechanism involved in the fungal infection on citrus,the function of SNF1 and FTR1 of A.alternata were characterized.In this study,we optimized the process of protoplast preparation in the tangerine pathotype of Alternaria alternata,and improved the genetic transformation system mediated by polyethylene glycol(PEG)-protoplast.Meanwhile,the function of SNF1 and FTR1 were characterized using gene knock-out and complementation strategy.The main results are as follows:1.An efficient method for protoplast isolation of Z7 strain of Alternaria alternata was established.The results showed that 36 h-old mycelia cultured in PDB medium was adequate to protoplast isolation.1% Kitalase was most beneficial for digesting the mycelia.The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5 h at 30?,100 rpm.Protoplasts were obtained at 4.36×108 /m L.In addition,0.7 M Na Cl was chosen as osmotic stabilizer in this study.PCR products were combined and transformed into fungal protoplasts prepared by above process from the Z7 strain using Ca Cl2 and PEG.Enough transformants were gotten,so the method achieved the desired effect..2.Split-marker was used to construct Aa SNF1 and Aa FTR1 gene knockout box PEG mediated protoplast transformation was carried out to gain gene knock-out mutants,the mutants were picked out after screening and verification by antibiotics,PCR and southern blot.The efficient homologous recombination mechanism in yeast was used to construct the complementation vector,and the complementary transformants were got after transformation.The complementary strains CP-S-2 and CP-F-2 were obtained and chosed for the follow-up tests.3.Aa SNF1 is required for utilization of un-prefered carbon source,vegetative growth,conidiation,maintaining the function of cell wall and membrane and virulence in A.alternata.Firstly,disruption of Aa SNF1 impacted vegetative growth,conidiation,conidial morphology and germination.?Aa Snf1 mutants showed a significant reduction in growth on different media,with fewer aerial mycelium and darker color compared to the wild type.The conidiation of the?Aa Snf1 was 70% lower than that of wild type,besides,condial diameter decreased and colour became lighter.Moreover,the condial germination was also significantly delayed.At 2 h post incubation on glass surfaces,the germination rate of the ?Aa Snf1 mutants was only about 5%whereas 30% of the wild type conidia germinated.The frequency of germination of the wild type reached around 90% at 8 h,when that of the ?Aa Snf1 strains showed 50%.Ultimately,most of the wild type condia germinated at 12 h,when the germination rate of the ?Aa Snf1 mutants were approximately 80%.Secondly,Aa SNF1 is required for fungal virulence.Disruption of Aa SNF1 resulted in a substantial reduction in pathogenicity,the ?Aa Snf1 mutants didn't or occasionally induced visible lesions.Thirdly,growth of the ?Aa Snf1 mutants was disturbed on MM media supplemented,respectively,with polygalacturonic acid,surcose and alcohol as the only carbon source.In addition,compared to the wild type,the ?Aa Snf1 showed stronger resistance to cell wall stress factor,such as SDS and CR.Complementally,disruption of Aa SNF1 didn't impact the osmotic and oxidative stress resistance of A.alternata.The complementation of Aa SNF1 return the phenotype of ?Aa Snf1 to the wild type level.4.Aa FTR1 expression was significantly reduced under iron-limited conditions,and the relative expression quantity was 32 times higher than that of the control.The disruption of Aa FTR1 also led to a reduction of intracellular iron content,which became more significantly under iron-limted conditions.However,the phenotypic analysis revealed that there is no significant difference between ?Aa Ftr1 and wild type in terms of colony phenotype,growth rate,conidiation and stress response.Although there is no difference in pathogenicity of mutant and wild type,the expression of Aa FTR1 was up-regulated during the primary stage of infection,and reached a peak at 48 h.Compared to the WT grown in PDB,the expression of Aa FTR1 was up-regulated 7 fold at 48 h post inoculation.