Functions In Ambient PH Adaptation And Pathogenesis Of The Colletotrichum Fructicola Transcription Factor PacC

Apple Glomerella leaf spot,mainly caused by Colletotrichum fructicola,is a fungal disease occuring in recent years in China.The disease causes leaf spots,defoliation and fruit drop before harvest,posting a great threat to apple production and causing serious economic loss.Fungi rely on pH sensing and responsive pathways to survive through complex pH conditions,in which the ZinC finger transcription factor PacC is a central component.Alkaline pH activates PacC,which further activates the transcription of some genes and inhibits other genes.Traditional apple bitter rot pathogens do not cause lesions on fruits at young stages,while C.fructicola can cause typical spot symptoms.Young fruits are more acidic than mature fruits,it is likely that C.fructicola adapts to acidic pH better than bitter rot pathogens,and such adaptation is important for Glomerella leaf spot disease development.In this study,we characterized the function of C.fructicola PacC gene,and determined the effects of its deletion on fungal growth,sporulation,pathogenicity and pH adaptability.Main results are listed as follows,1)This study used split PCR method to construct gene knockout fragments containing hph selection marker,transformants resistant to hygromycin B were obtained by PEG-mediated protoplast transformation.PCR assays were performed,and four PacC gene knockout mutants were obtained.2)Differences in biological characteristics and pathogenicity between mutants and wild type were systematically studied.The results showed that the growth rate,the rates of conidial germination and appressorium formation were significantly decreased in the mutants,which indicate that PacC has great influence on the conidial germination,appressorium formation and growth rate.Pathogenicity tests were performed with Gala leaves or Gala fruits without or with wounding.On leaves,significant virulence reduction of mutants was observed with both unwounded and wounded inoculations;on fruits,virulence reduction was observed with unwounded inoculation but not wounded inoculation.Complementary transformants of PacC gene knock out mutants were obtained by constructing a complementary vector,G418-based selection and PCR screening.The complementary transformants were restored to the wild-type state in colony morphology and growth rate,the rate of conidial germination and appressorium formation,the pathogenicity toward Gala leaves.Those indicate that the biological and pathogenicity changes of the knockout mutants are due to the deletion of the PacC gene.3)Mutants and wild type were inoculated onto PDA medium buffered at different pHs,the results showed that there was no significant growth difference between mutants and wild type at pH3;for pH4 to pH6,growths of mutants were significantly slower than wild type,and differences in growth rates increased as pH became higher,indicating that PacC is functionally important for growth in an acidic environment.At pH8,growth rates of mutants were drastically decreased;pH9 completely inhibited mutant growth whereas wild type could still grow at a slow rate,indicating that PacC is essential for fungal growth at alkaline pH conditions.In summary,PacC gene affects the growth,conidial germination,appressorium formation,and pathogenicity of C.fructicola.This gene is related to C.fructicola growth under acidic and alkaline conditions.