Study On The Function Of Arthrobotrys Oligosporus Calcium/calmodulin-dependent Protein Kinases (CaMKs)

Arthrobotrys oligospora,a typical nematode-trapping fungus,can capture and infect nematodes via forming special three-dimensional networks to acquire necessary nutrition,which is considered as a model fungus to explore the relationship between fungi and nematodes.Ca2+/calmodulin-dependent kinases are important multifunctional signal proteins,locate in the downstream of G protein signal pathway,which response to various extracellular stimuli to regulate multiple biological processes.In this study,five CaMK proteins are identified and functionally characterized in A.oligospora based on the gene knockout technology,phenotypic analysis,environmental stress tolerance,real-time PCR analysis and so on.The main experimental results:1.Functional domains,physicochemical properties and phylogenetic analyses of five CaMKs in A.oligospora The conserved domains were predicted by InterProScan,five CaMKs from A.oligospora contain two conserved domains,namely,a protein kinase-like domain and a protein kinase domain,and several functional sites,such as a protein kinase ATP binding site(IPR17441),a serine/threonine-protein kinase active site(IPR008271),and a conserved activation loop(A-loop).Physicochemical properties analyses revealed that but CaMKKC2 gene encodes a polypeptide of 1251 amino acid residues with a large molecular weight of 128.7 kDa,the other four CaMKs were found to possess similar amino acid residues and molecular weights,all of which were lower than those of CaMKK C2.Meanwhile,no significant difference was found in the pI of the five CaMKs.Cluster analysis shows that five CaMKs are divided into two different clades(I and II).Among them,clade I is CaM kinase,and clade II is CaM kinase kinase.2.Knock-out of five CaMKs genes and comparison of their phenotypes and environmental stress tolerance Knockout vectors of the five CaMKs genes were constructed and transformed into A.oligospora protoplast via CaCl2-PEG mediated transformation,and the five mutants of CaMK genes were successfully obtained.Then phenotypic traits of five mutants were compared with the wide type(WT)strain in growth rate,colonial morphology,chemical stress,and environmental stress(thermotolerance and UV radiation).Our results showed that the growth rate of five mutants were slower than the WT strain,and the colony morphology of ACaMKK C2 differed from that of the WT and other mutants on TYGA medium,with ?CaMKK C2 exhibiting compact,somewhat small aerial hyphae.Five mutants showed growth defects on TG medium supplemented with different concentrations of chemicals(H2O2,menadione,SDS,and Congo red),with variant sensitivity to these chemicals.Moreover,the environmental stress tolerance experiments revealed that five CaMKs to varying degrees were involved in regulating thermotolerance and UV resistence in A.oligospora.3.Analysis of conidial yield,trap formation and pathogenicity in five ACaMKs mutants Compared with the WT strain,the five ?CaMKs mutants exhibited significantly reduced in sporulation.In order to probe the mechanism of CaMKs regulate conidiation,the hyphae of the WT and five mutants were collected after incubated on TYGA plates for 3 d,5 d and 7 d for RNA extraction.Total RNA of all samples were extracted and transcribed into cDNA.Transcription levels of 12 sporulation-related genes in A.oligospora were determined by RT-PCR with specific paired primers.The transcription levels of FlbA,FlbC,MedA,NsdD,Pka,VelB,VosA,and VeA were down-regulated in the five mutants at each time point.In addition,the traps number and captured nematodes of five mutants were slower than the WT strain,and the trap formation was retarded in the ACaMK B mutant.In general,above results suggested that CaMKs were involved in the biological processes of conidiation and pathogenicity in A.oligospora.Innovations of this paper:1.The functions of CaMKs were identified in the nematode-trapping fungi for the first time.Five CaMKs genes were successfully knocked out in A.oligospora,and they involved in the regulation of multiple biological processes,such as growth,conidiation,environmental stress tolerance,trap formation,and pathogenicity.2.Comparison of thermotolerance of the ACaMKs mutants and WT strain,suggested that CaMKs involved in regulation of heat shock,which in turn influenced hyphae growth and conidiation in A.oligospora.3.Comparision of UV resistance between the OCaMKs mutants and WT strain,our results showed that the OCaMKs mutants were more sensitive than the WT strain to UV radiation in A.oligospora,with ?CaMKA being significantly sensitive.