The Effects Of Two Splice Isoforms Of FpNcb2 On Nuclear Location Of FpNcb2 And Virulence Of Fusarium Pseudograminearum

Fusarium crown rot(FCR)caused by Fusarium pseudograminearum is a soil-borne disease that occurs widely worldwide and severely affects wheat yield and quality.In recent years,FCR has spread rapidly in major wheat producing areas in China,and there is a lack of effective disease-resistant wheat varieties in production,so fungicides are the most important strategy to control FCR.Inappropriate use of fungicides with the same mode of action can lead to resistance,and the detection of resistance related genes in F.pseudograminearum may provide reference for the assessment of the risk of drug resistance.Ncb2 is a component of the NC2 heterodimer in eukaryotes and is a key regulator of fungal resistance to azole fungicides,but its role in phytopathogenic fungi remains unknown.In this study,the biological function and regulatory mechanism of FpNcb2 were systematically analyzed,which may provide a theoretical basis for analyzing the mechanism of drug resistance and pathogenicity in F.pseudograminearum.The main study findings are as follows.FpNcb2 is involved in drug resistance and pathogenicity of F.pseudograminearum.The FpNcb2 deletion mutant(ΔFpNcb2)was obtained by PEG-mediated genetic transformation,and the growth rate of ΔFpNcb2 was significantly reduced compared with the wild type and failed to produce conidium,the ΔFpNcb2 mutant showed significantly reduced tolerance to tebuconazole and enhanced tolerance to phenamacril and cyclobutyifluram,and qRT-PCR analysis revealed that the deletion of FpNcb2 did not affect the transcription of tebuconazole and phenamacril target genes.Moreover,we found thatΔFpNcb2 was unable to infect the wheat coleoptiles and barley leaves under wounded and non-wounded treatment.The above results indicate that FpNcb2 also regulates multiple drugs resistance,and is involved in growth,conidiation and pathogenicity of F.pseudograminearum.The two splicing isoforms of FpNcb2 are associated with the nuclear accumulation of FpNcb2.Alternative splicing of eukaryotic transcripts is a ubiquitous regulatory mechanism that contributes to the expansion of proteome complexity by generating multiple transcript isoforms from a single gene.In this study,two mRNA splicing isoforms of FpNcb2 were identified by 5’ race analysis,producing two protein isoforms,the FpNcb2IsoA and FpNcb2IsoB,with different N terminus.As a CCAAT binding protein,FpNcb2 predominantly localized to the nucleus in F.pseudograminearum.However,the fluorescent signals of both FpNcb2IsoA-GFP and FpNcb2IsoB-GFP were diffused in hypha,and localised to the punctate in conidia.In germlings,FpNcb2IsoA-GFP localised to the nucleus,while FpNcb2IsoB-GFP still localised to particles in the cytoplasm(Fig.3B).When FpNcb2IsoA-GFP and FpNcb2IsoB-GFP were concurrently expressedin F.pseudograminearum,the GFP signals were predominantly re-gathered to the nucleus.As expected,we confirmed FpNcb2IsoA interacts with FpNcb2IsoB in F.pseudograminearum by Co-IP assays.The results suggest a hypothetical link of the interaction between two isoforms of FpNcb2 and the nuclear location of FpNcb2.The two isoforms of FpNcb2 have different effects on the pathogenicity of F.pseudograminearum.Using genetic transformation methods,we transformed the FpNcb2IsoA and FpNcb2IsoB into the FpNcb2 deletion mutant,and generated the FpNcb2IsoA and FpNcb2IsoB strains.Expression of each isoform could fully complement the defects ofΔFpNcb2 in growth,conidiation and sensitivity to fungicides.Compared to the WT and complemented strains,FpNcb2IsoA revealed equal pathogenicity,whereas FpNcb2IsoA strain caused reduced FCR.Furthermore,we found that FpNcb2IsoA and FpNcb2IsoB showed different germination rates which may be related with the subcellular localization of the two isoforms during conidia germination.The FpNcb2 loss in F.pseudograminearum enhanced sensitivity to oxidative stress,cell membrane and cell wall stresses,and each isoform of FpNcb2 successfully rescued the defects of the corresponding mutant to these abiotic stresses.Using ChIP-seq and qRT-PCR analysis,we found that FpNcb2 regulates the expression of various efflux pump antibiotic resistance protein and chitinases in F.pseudograminearum,which might contribute to drug resistance and pathogenicity of F.pseudograminearum.In summary,FpNcb2 is involved in the growth,conidiation,drug resistance,and pathogenicity of F.pseudograminearum,and two isoforms generated by alternativemRNA splicing of FpNcb2 associated with nucleus accumulation of FpNcb2 and the pathogenicity of F.pseudograminearum.The results will aid the understanding of the regulatory mechanisms of pathogenicity in F.pseudograminearum,and provide scientific foundation for efficient disease control strategies.