Resequencing And Transcriptome Analysis Of Alternaria Alternata Croftonweed Pathotype Mutant

Alternaria alternata(Fr.)Keissler is a natural pathogen of invasive weed Croftonweed(Ageratina adenophora Spreng.).Alternaria alternata can cause leaf spot on leaf of croftonweed and kill seedling of croftonweed.We previously found that TeA toxin of A.alternaria had strong herbicidal activity.The main target of TeA is D1 protein in chloroplast of plant cell.TeA can bind D1 protein and block photosynthetic electron transfer,causing active oxygen bursts in chloroplast and eventually lead to cell necrosis.TeA has high herbicide activity,simple structure and fast action.It has potential for broad-spectrum herbicidal activity on monocotyledonous and Dicotyledonous weeds and become a multi-function bio-herbicide.However,the A.alternaria isolate produce low amount TeA,which is difficult to ferment large quantities of TeA.Therefore,understanding mechanism of the TeA production of A.alternaria and then modifying the strain become urgent.A non-pathogenic mutant was obtained by restriction endonuclease-mediated integration(REMI).We have previously determined that the histidine phosphatase family HP001 gene was inserted using Anchored PCR,and we also proved that HP001 affects the fus3 MAPK signal pathway by affecting the Gβ gene of G protein signaling pathway.The A.alternaria mutant has been partially restored pathogenicity by adding the wild-type A.alternata filtrate or TeA which themselves could not cause the lesion,thus demonstrating that TeA and HP001 are two virulence factors of A.alternaria.This paper mainly focuses on the relationship between the two strains of A.alternaria,using high-throughput sequencing to obtain whole genome sequences and transcriptome sequences.Through annotation and analysis of these results we can understand biological synthesis process of TeA toxin,pathogenic mechanism of the pathogen and provide with the necessary information for subsequent specific gene modified and disease occurrence studies.Our results are as follow:The mutant is largely different from wildtype through genome resequencing.the mutant genome has size of 33.18Mb while genome of wild-type A.alternaria is 33.09Mb,and mutant has more repeat sequence.Via comparison with the wild type,we have found 191 insertions in the mutant which affecting 241 genes.After analyzing these inserted genes and their nearby genes,we found some oxidoreductases,dehydrogenases,glutathione-s-transferase,pigment-related,α-amino acid related genes,iron binding genes,phosphotransferase activity and dephosphorylation items among them.dephosphorylation item have HP001 gene within.Sampling for transcriptome was carried out during the growth process of A.alternaria.Analysis of transcriptome results is consistent with previous studies:Mu is less active than WT of biological activity and metabolic activity,so it grows looser in liquid medium;MAPK pathway Slt2 gene is down-regulated,so cell wall synthesis is suppressed and forming defective cell walls.The Hog1 gene regulates the metabolism of TeA and salt tolerance of the Mu strain.At the same time,the AP1 gene that regulates ROS degradation in Mu is down-regulated and some oxidoreductases are also down-regulated in the inserted gene and surrounding regions of insertion sites.These factors together make it difficult for Mu to cope with intracellular and extracellular ROS and meanwhile may contribute to less secondary metabolites.Therefore,Mu strain accumulate high reactive oxygen content in its mycelium.Excessive ROS make Mu strain unable to maintain metabolism during growth,destroy cell components,and induce programmed cell death.In addition,expression of melanin synthesis gene BRM2 which may be regulated by LaeA regulator is down-regulated in the mutant and thus Mu strain appear grayish color.On the second day within growth process,expression changes of the transcriptome revealed that Mu has 1840 down-regulation genes and 1559 up-regulation genes.Differentially expressed genes are mainly enriched in oxidoreductase,transmembrane transport,iron binding,biological processes and metabolic processes of GO terms.On the fourth day,Mu have 1517 down-regulation genes,and up-regulation 1624 genes.The oxidoreductase-relative genes,catalytic genes and iron-binding genes are still up-regulated.On the sixth day,Mu have 799 down-regulation genes and 1036 up-regulation genes.The differential genes between WT and Mu strain is less than before,which may due to nutrient depletion in liquid medium and aged strains,differential gene expression begins to decline.Additionally,we also found that a part of Mu genes associated with pathogenicity of A.alternata is downregulated,and the expression of most of the cell wall degradation-related enzymes genes are down-regulated.At the same time,the gene cluster controlling TeA synthesis in the secondary metabolic gene cluster is down-regulated,so mutant cannot produce enough TeA toxin.All these factors contribute to pathogenicity loss of Mutant.11 genes in total was found after filtering significantly down-regulated genes in 241 inserted genes.Compared to previously studied HP001 gene,the effects of other genes are limited.We also found that genes related to iron-ion binding in the peripheral region of the insertion site are abundant,which may be related to the sensitivity of Mu to ROS.To further validate the differential gene expression in A.alternata,we use real-time PCR to verify core genes of the pathway.The TeA synthesis gene TAS1 and its putative transportor gene MFS highly express during initial growth phase.While expression of them rapidly declines when nutrition begins to decline.LaeA gene positively regulate TeA synthesis while Hog1 gene negatively regulate TeA synthesis.At the same time,UPLC-MS was used to confirm that HP001 gene has a important role in the production of A.alternata.We found that the wild type of A.alternata had higher concentration of TeA than mutant during whole growth period,and the ratio of WT to mutant are higher on the third and fourth days,and ratios at these time are 200.37 and 203.96 times respectively.The HP001 complementation strain M1 produces higher toxin production than the mutant through whole growth period,and on the fourth day,the ratio of M1 to Mu is the highest,which is 245.60 times more TeA than Mutant.HP001 overexpressing strain W1 accumulates toxins faster than the wild type during initial growth phase.In summary,mutant produces less TeA toxins due to loss of the HP001 gene,and thus is insufficient to complete infection and losing toxicity.