Studies On The Functions And Mechanisms Of Poly (ADP-ribose) Polymerase FonPARP1 In Pathogenicity Of Fusarium Oxysporum F.sp.niveum On Watermelon

Watermelon Fusarium wilt,caused by the soil-borne pathogenic fungus Fusarium oxysporum f.sp.niveum(Fon),is a devastating disease that greatly influences the yield and quality of watermelons and hinders the development of watermelon industry in China.However,knowledge on the molecular network governing the pathogenicity in Fon is limited.Protein poly(ADP-ribosyl)ation(PARylation)is a reversible post-translational modification widely present in almost all organisms.PARylation is catalyzed by poly(ADP-ribose)polymerases(PARPs),which transfer ADP-ribose moieties from NAD~+to target proteins,but mainly hydrolyzed by poly(ADP-ribose)glycohydrolase(PARG)that removes PAR chains or individual ADP-ribose moieties from substrates.This study aimed to investigate the function and mechanism of PARylation,mediated by PARPs and PARG,in the pathogenicity of Fon using a combination of biochemistry,mass spectrometry analysis,molecular biology,and plant pathology.A single Fon PARP1 gene and one Fon PARG1 gene were identified in Fon genome.Fon PARP1 possesses multiple conserved domains,including BRCT,WGR,PARP1＿reg,and PARP1 domains,while Fon PARG1 contains a single conserved PARG＿cat domain.Targeted deletion mutantsΔFon PARP1 andΔFon PARG1 were generated by the homologous recombination strategy to investigate the biological functions of Fon PARP1 and Fon PARG1 in Fon.Phenotypic analyses showed that Fon PARP1 was involved in regulating mycelial growth under nutrient-deficient conditions and abiotic stress responses of Fon,whereas Fon PARG1 participated in abiotic stress responses.Deletion of Fon PARP1 impaired the pathogenicity of Fon by affecting the invasive growth within watermelon plants,whereas Fon PARG1 was dispensable for Fon pathogenicity.Fon PARP1 was localized in nucleus and possessed active PARP activity with self-PARylation ability in in vitro PARylation assays.Either the conserved domains,e.g.,BRCT,WGR,PARP1＿reg,and PARP1 domains,or the conserved active residue E729 was essential for the enzymatic activity and the functions of Fon PARP1 in abiotic stress responses and pathogenicity of Fon.Fon PARG1interacted with Fon PARP1 and exhibited PARG activity,leading to hydrolysis of the self-PARylation on Fon PARP1.A total of 53 potential Fon PARP1-interacting proteins were identified,among which Fon Kin4,a mitosis-related serine/threonine protein kinase,was shortlisted.The interaction between Fon Kin4 and Fon PARP1 was validated by yeast two-hybrid,pull-down,and co-immunoprecipitation assays.Targeted disruption mutantΔFon Kin4 was constructed using the homologous recombination strategy.Phenotypic analyses revealed thatΔFon Kin4 exhibited defects in mycelial growth,asexual reproduction,macroconidial morphology,and abiotic stress responses.Importantly,ΔFon Kin4impaired the pathogenicity on watermelon through influencing the invasive growth within watermelon plants.Fon Kin4,containing the conserved S＿TKc domain,was localized in septum and harbored serine/threonine protein kinase activity,which enabled its self-phosphorylation in vitro.Either the conserved S＿TKc domain or the conserved active residue T462 was essential for its kinase activity and functioned in basic biological processes,abiotic stress responses,and pathogenicity of Fon.Fon Kin4unidirectionally phosphorylated Fon PARP1 in vitro,while Fon PARP1 did not PARylate Fon Kin4.Furthermore,Fon Kin4-mediated phosphorylation promoted the PARP activity of Fon PARP1.A direct interaction of Fon PARP1 with the protein disulfide isomerase Fon Pdi1was detected.The BRCT domain-containing N-terminal of Fon PARP1 was the specific region responsible for its association with Fon Pdi1.However,Fon PARP1 did not interact with other Fon Pdis.Fon PARP1 specifically PARylated Fon Pdi1 in vitro and in vivo.Fon PARG1 also interacted with Fon Pdi1 and hydrolyzed the Fon PARP1-mediated PAR on Fon Pdi1.A total of 21 PARylated sites,including 13 glutamic acid residues and8 aspartic acid residues,were identified in Fon Pdi1,with 13 glutamic acid residues being critical for Fon PARP1-mediated PARylation on Fon Pdi1.The PARylation level of Fon Pdi1 not only impacted its interaction with Fon PARP1,but also influenced its PDI activity and function in the pathogenicity of Fon.Furthermore,Fon PARP1 and Fon Pdi1 PARylation were also involved in regulating ER homeostasis and function,affecting the secretion of extracellular pectinases in Fon.In conclusion,this study demonstrates the critical role of Fon PARP1-catalyzed PARylation in the pathogenicity of Fon.The PARP activity of Fon PARP1 is controlled by an upstream protein kinase Fon Kin4,which is also essential for Fon pathogenicity.Fon PARP1 PARylates its substrate Fon Pdi1 to modulate the PDI activity,which impacts ER function and pathogenicity of Fon.These findings provide new insights in understanding the molecular network underlying the pathogenicity of Fon.