Functions And Molecular Mechanisms Of PdCERKs And PdCCaMK1 In Poplar-Mycorrhizal Symbiosis

Mycorrhizae are mutualistic symbiosis formed between plants and mycorrhizal fungi.Ectomycorrhizae(Ec M)and arbuscular mycorrhizae(AM)are the most extensively studied types of mycorrhizae.Ectomycorrhizal fungi can establish symbiotic relationships with various woody plants,which is of great significance in maintaining the stability of forest ecosystems.However,the molecular mechanism of the symbiotic relationship between woody plants and Ec M fungi is not clear,thereby,impeding the application of ectomycorrhizal fungi in forest production.Significant process has been made in the molecular mechanisms of herbaceous plants and AM fungi symbiosis including the widely accepted common symbiosis signaling pathway(CSSP).In this pathway,the recognition and transduction of arbuscular mycorrhizal symbiosis signals rely on the involvement of key components,including plant cell membrane-localized CERK1(Chitin elicitor receptor kinase)and cell nuclear-localized CCaMK(Calcium/calmodulin-dependent protein kinase).The roles of CERK1 and CCaMK in regulating the establishment of symbiotic relationships between woody plants and arbuscular/ecto-mycorrhizal fungi remains unclear.As one of the most widely planted fast-growing tree species,poplar can establish symbiotic relationships with both arbuscular mycorrhizal fungi and ectomycorrhizal fungi,and is a typical species of both endo-and ectomycorrhizal trees.In this study,“Nanlin 895”poplar(Populus deltoides × Populus euramericana cv.)was used as the woody plant material,Laccaria bicolor and Rhizophagus irregularis were used as the model strains of ectomycorrhizal and arbuscular mycorrhizal fungi,respectively.Symbiotic systems of poplarL.bicolor and poplar-R.irregularis were established.Through various experimental methods,including genetics,cell biology,and biochemistry,the functions and mechanisms of CERK1 and CCaMK in poplar-mycorrhizal symbiosis were deeply investigated.The results are as follows:(1)Using CRISPR/Cas9 gene editing technology,different types of mutant plants have been obtained,including Pdcerk1 single mutants(cerk16 and cerk34),Pdcerk1/2 double mutants(cerk7 and cerk57),Pdcerk2/3 double mutants(cerk65 and cerk117),Pdcerk1/2/3triple mutants(cerk26 and cerk33),and Pdccamk1 single mutants(Pdccamk1-1 and Pdccamk1-2).(2)To investigate the function of PdCERKs in the symbiotic association between the poplar and arbuscular mycorrhizal fungi,the symbiotic structures of arbuscular mycorrhizal fungi were observed in wild-type and Pdcerk1/2/3 mutants(cerk26 and cerk33).Microscopic observation showed that continuous arbuscular structures and internal hyphae were observed in the wild-type plants,while arbuscular structures and internal hyphae were only occasionally observed in the cerk26 and cerk33 mutants.Compared to the wild-type plants,the colonization rate of arbuscular mycorrhizal fungi in the cerk26 and cerk33 mutant plants reduced by 78.8%and 80%,respectively,Transcriptome sequencing and quantitative real-time PCR analysis were performed on wild-type and cerk26 mutants inoculated with arbuscular mycorrhizal fungi.It found that the expression of marker genes(AM3,AM14,PT4,HA,and RAM1)in arbuscular mycorrhizal symbiosis was reduced in the cerk26 mutant plants.These results indicates that PdCERKs are involved in regulating the symbiotic relationship between the poplar and arbuscular mycorrhizal fungi.(3)To investigate the function of PdCCaMK1 in the symbiotic association between the poplar and arbuscular mycorrhizal fungi,the symbiotic structures of arbuscular mycorrhizal fungi were observed in wild-type,ccamk1-1,and ccamk1-2 mutants.Microscopic observation revealed abundant arbuscular structures in the wild-type plants,whereas the ccamk1-1 and ccamk1-2 mutants exhibited a complete absence of arbuscular structures,with only occasional presence of internal hyphae.Compared to the wild-type plants,the colonization rate of arbuscular mycorrhizal fungi in the ccamk1-1 and ccamk1-2 mutant plants reduced by 99.7%and 98.2%,respectively.Transcriptome sequencing and quantitative PCR analysis were conducted on wild-type and ccamk1-1 mutant plants after inoculation with arbuscular mycorrhizal fungi.It was discovered that the expression of marker genes(AM3,AM14,PT4,HA,and RAM1)associated with arbuscular mycorrhizal symbiosis was reduced in the ccamk1-1 mutant plants.These results indicates that PdCCaMK1 is involved in regulating the symbiotic relationship between poplar and arbuscular mycorrhizal fungi.(4)To investigate the function of PdCERKs in the symbiotic association between the poplar and ectomycorrhizal fungi,the colonization rate of the ectomycorrhizal fungi was calculated in wild-type,cerk26,and cerk33 mutants.Compared to the wild-type plants,the colonization rate of ectomycorrhizal fungi in the cerk26 and cerk33 mutant plants reduced by87.4% and 93.5%,respectively.To further observe the structure of ectomycorrhiza,colonized root tips were sectioned,and observed by confocal laser scanning microscope.Wild-type plants exhibited thick mantle and dense Hartig net structures.However,in the cerk26 and cerk33 mutants,there was a reduction in the symbiotic structures,specifically the Hartig net.The Hartig net in the cerk26 and cerk33 mutants decreased by 78.4% and 84%,respectively.Additionally,the thickness of the mantle was also reduced in these mutants,with reductions of 55.8% and 63.9%,compared to the wild-type plants.These results indicates that PdCERKs are involved in regulating the symbiosis between the poplar and ectomycorrhizal fungi.(5)Through yeast two hybrid,Pull Down,and Co-immunoprecipitation(Co-IP)assays,it was discovered that the interaction of between PdCERK1 and the lectin receptor-like kinase PdLec RLK1.PdCERK1 exhibited kinase activity,and the kinase activity was lost after mutation of the K335 site of PdCERK1 by in vitro phosphorylation experiments.Transient expression of tagged PdCERK1 and PdLec RLK1 fusion proteins in tobacco(Nicotiana benthamiana)leaves was used to perform semi-in vitro phosphorylation experiments.The activation of PdCERK1 kinase by ectomycorrhizal fungal secretions was observed.When PdCERK1 and PdLec RLK1 were co-expressed in N.benthamiana leaves,the activation of PdCERK1 by the secretions was more pronounced.These results suggested that the regulation of PdCERK1 kinase activity by ectomycorrhizal fungi secretions depends on PdLec RLK1.(6)Through yeast two-hybrid,Pull Down,and Co-IP assays,it was discovered that the interaction of between PdCERK1 and the effector protein Mi SSP8(Mycorrhiza-induced small secreted protein 8)secreted by ectomycorrhizal fungi.In vitro phosphorylation experiments showed that Mi SSP8 inhibits the phosphorylation level of PdCERK1.In summary,this study investigated the functions of PdCERKs and PdCCaMK1 in poplar-mycorrhizal symbiosis,particularly in ectomycorrhizal symbiosis,and elucidated the molecular mechanism of PdLec RLK1-PdCERK1-Mi SSP8 in regulating the process of ectomycorrhizal symbiosis in poplar.These findings contribute to a better understanding of the mycorrhizal symbiotic process in woody plants,and provide the theoretical basis for the application of mycorrhizal fungi in forestry production.