Molecular Mechanisms Of Colletotrichum Higginsianum To Arabidopsis

The hemibiotrophic fungus Colletotrichum higginsianum is the causal agent of anthracnose disease of cruciferous plants including many cultivated species of Brassica and Raphanus as well as the model plant Arabidopsis thaliana.The spores of C.higginsianum adhere to the plant surface and germinate in proparate conditions,then form appresoria to penetrate the host tissue and then cause disease.The C.higginsianum-A.thaliana pathosystem,in which both partners can be genetically manipulated and genome sequences are publicly available,provides a well-supported model system to examine hemibiotrophic fungal-plant interactions.It is thus necessary to examine the genetic control of C.higginsianum for improving our understanding of molecular fungal pathogenic mechanisms,and also or developing novel biocontrol strategy for this important anthracnose disease.We examine the Ch Rgf and Ch Acs gene function in C.higginsianum and the subcellular reaction of host A.thaliana upon penetration by C.higginsianum.The main research result were as follows:We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation(ATMT)to isolate pathogenicity mutants of C.higginsianum.From a collection of 2000 insertion mutants we isolated 7 mutants with reduced symptoms.Southern blot analysis indicated that the mutants Ch-1-G256,Ch-1-G281,Ch-1-G310,Ch-1-G323,Ch-1-G668 and Ch-1-E240 harbored single-site T-DNA integrations,while Ch-1-G090 harbored two T-DNA insertions.Border flanking sequences of T-DNAs from these mutants were recovered by inverse polymerase chain reaction(PCR).Sequence analyses revealed that single T-DNA insertions in mutant Ch-1-G281 targeted a gene coding Ras GEF.The two T-DNA insertion sites in mutant Ch-1-G090 had insertions upstream of genes for a hypothetical protein and a RNA processing enzyme.A single T-DNA insertions in mutant Ch-1-G256 targeted a gene coding lactose permease.The single T-DNA insertions in mutant Ch-1-G310 and Ch-1-G668 targeted gene coding hypothetical protein.A single TDNA insertions in mutant Ch-1-G323 targeted a gene coding serine/threonine-protein kinase cot-1.The single T-DNA insertions in mutant Ch-1-E240 targeted a gene coding Acetyl-Co A enzayme.The Ch Rgf gene encoding a Ras guanine-nucleotide exchange factor protein was identified by screening T-DNA insertion mutants generated from Agrobacterium tumefaciens-mediated transformation(ATMT)that were unable to cause disease on the host Arabidopsis thaliana.Targeted gene deletion of Ch Rgf resulted in a null mutant(ΔChrgf-42)with defects in vegetative growth,hyphal morphology and conidiation,and poor surface attachment and low germination on hydrophobic surfaces,but there were no apparent differences in appressorial turgor pressure between the wild-type and the mutant.The conidia of the mutant were unable to geminate on attached Arabidopsis leaves,and did not cause any disease symptoms.Intracellular c AMP levels in the ΔChrgf mutant were lower than that of the wild-type.Our results suggest that Ch Rgf is a key regulator in response to salt and osmotic stresses in C.higginsianum,and indicate that it is involved in fungal pathogenicity.This gene seems to act as an important modulator upstream of several distinct signaling pathways that are involved in regulating vegetative growth,conidiation,infection-related structure development and stress responses of C.higginsianum.The Ch Acs1 gene encoding Acetyl-Co A synthase was identified by screening T-DNA insertion mutants generated from Agrobacterium tumefaciens-mediated transformation(ATMT)that having difficiency in causing disease on the host Arabidopsis thaliana.Targeted gene deletion of Ch Acs1 and Ch Acs2 resulted in null mutant with no affects in vegetative growth,hyphal morphology and conidiation,and the germination on hydrophobic surfaces is almost the same with the wild-type.The Ch Acs1 deletion resulted in a defect in utilize unfermentable carbon source such as acetate,ethanol and acetaldehyde.The Ch Acs1-dependent acetyl-Co A production might be required for variable physiological processes in addition to lipid.The conidia of the Ch Acs1 mutant has delayed disease symptoms on Arabidopsis leaves,the conidia germination,appressoria formation and the primary hyphae formation were all delayed in the Ch Acs1 mutant.Subcellular localization showed that Ch Acs1 and Ch Acs2 were localized in cytosol.RNA-seq data showed that Ch Acs1 gene involved in many cellular progress such as ethanol metabolism,TCA and glyoxlate cycles.Our results suggest that Ch Acs1 is a key component of the pyruvateacetaldehyde-acetate pathway,this fermentation process might have crucial roles in various physiological processes for filamentous fungi.We applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with the primary hyphae infection stage in Arabidopsis.Plasma membrane-resident proteins differentially localised to the biotrophic interface.Likewise,plasma membrane-lipid proteins had a different pattern of localization to the biotrophic interface which suggesting a role of tonoplast involving in the interface interaction.The latter is supported by the tonoplast marker closely surrounded the biotrophic hyphae.Moreover,a correlation was observed between the collapse of vacuoles and transition of primary hyphae to the thin secondary hyphae in the first invaded cell.Furthermore,endosomal compartments surrounded the biotrophic hyphae revealed that the interface of biotrophic hyphae had modified endosomal characteristics.The ARA7/RABF2 b was specially localized to the biotrophic interface.These findings provide a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes of this particular hemibiotrophic fungus Colletotrichum higginsianum.