Molecular Mechanism Of ClNPS6 And ClFtr1 Regulating Pathogenicity In Curvularia Lunata

Curvalaria Leaf Spot is one of the major leaf diseases in maize,which caused by Curvularia lunata（Walk）Boed..At present,researchs on C.lunata mainly focus on toxins,melanin and cell wall degrading enzymes.It has been shown that iron is an indispensable element for pathogenicity.Non-ribosomal peptide synthase 6（NPS6）and Fe transporter permease（Ftr1）are the key genes respectively regulating siderophore absorption（SIA）and reduced iron absorption（RIA）.This study reveals the mechanism of NPS6 and Ftr1 affecting pathogenicity and analyses transcriptomics to find other pathways required for pathogenicity.It is foundation for studying the relationship between ClFtr1,ClNPS6 and pathogenicity of C.lunata,and explain corn pathogenic mechanism of Curvularia leaf spot from the point of iron metabolic,to enrich machanism of molecular pathogenesis.The main results are as following:（1）To determine the relationship between ClNRPS and pathogenicity of C.lunata.Six NRPS genes（ClNPS1,ClNPS2,ClNPS4,ClNPS6,ClNPS8 and Cl NPS10）were obtained by PCR from the genome of C.lunata,and the characters of genes were predicted and analyzed by bioinformatics.ClNRPS gene knockout vectors were constructed,and transformants were obtained by Agrobacterium tumefaciens-mediated transformation（ATMT）.The results showed that pathogenicity of△Clnps6 was obviously decreased.（2）To determine iron is an essential nutrient for promoted the pathogenicity of C.lunata.The influence of iron was determined by investigating the biological characteristics,pathogenicity,toxin and cell wall degrading enzymes（CWDEs）activity of C.lunata.The growth and spore production of C.lunata were not affected by different Fe³⁺concentrations（0-80μM）.Spore germination rate was the highest after 8 h under 40μM Fe³⁺concentration,and 35%higher than those of under Fe³⁺-limited conditions,consistent with germ tube length.Exogenous application of Fe ³⁺（more than 20μM）significantly accelerated the development of Curvularia leaf spot disease.Toxin assays results showed that it had a certain pathogenic effect on leaves,however,it was independent of iron concentration.With the increase of Fe³⁺concentration,CWDEs activity also increased.Polygalacturonase（PG）played a role in the early stages of infection.Cellulase（Cx）,Pectin methyl-galacturonase（PMG）,Polygalacuronic acid trans-eliminase（PGTE）,Pectin methyl trans-eliminase（PMTE）played a part after inoculation of 72 h.In addition,the CWDEs activity of C.lunata were not affected by different Fe³⁺concentrations.Thus,we concluded that Fe³⁺promoted development and improve pathogenicity of C.lunata.（3）SIA and RIA regulate the pathogenicity of C.lunata under low iron conditions.q-PCR was carried out to analyze ClNPS6 and ClFtr1,ClNPS6 and ClFtr1 gene were upregulated under iron-depleted conditions.The knockout,complementation and double deletion transformants were obtained.ClNPS6 and ClFtr1 konckout and complementary vectors were constructed,transformants were obtained by ATMT.Under different concentration of Fe³⁺,the growth and pathogenicity of knockout and complementary transformants were determined.Compared with the wild type,there was no significant difference between knockout and complementary transformants in the growth rate,spore production and appressorium,but the spore germination of the knockout transformants decreased obviously,and the complementary transforming recovered.The pathogenicity of transformants was significantly decreased,exogenous iron restores the virulence of the transformants.The pathogenicity of complementary transformants and wild types were not significantly different.Sensitivity detection showed that the transformants were sensitive to hydrogen peroxide.（4）To identify the transporter related genes in the SIA affecting the pathogenicity of C.lunata.SIA pathway maintaining the pathogenicity of C.lunata was verified by analysis the functional of membrane transporter protein.ClFip1p,ClFip3p and ClSit1p were downregulated in△Clnps6,it indicated that the transport system of the membrane was restrained after the restriction of siderophore pathway.ClFip1p,ClFip3p and ClSit1p knockout vectors were constructed,transformants were obtained by ATMT.The growth and pathogenicity of transformants were determined.Compared with the wild type,there was no significant difference with transformants in the growth rate and spore production,but the spore germination and appressorium of the transformants decreased obviously.The pathogenicity of transformants was significantly decreased,exogenous iron restores the virulence of the knockout transformants.The above results indicated that ClNPS6 regulates the biosynthesis of siderophores,and transporter related genes affect the transport of siderophore-iron complexes,both of which were critical for the siderophores absorption pathway.（5）To clear the ClFtr1 regulating the appressorium formation and ClNPS6 regulating the activity of pathogenic factors for the pathogenicity of C.lunata.Compared with wild-type strains,there were 23 down-regulated genes were involved in the formation of appressorium,secondary metabolism,and signaling.Analysis the expression of ClNPS6 and ClFtr1 in the process of infection showed that the ClFtr1 was strongly expressed at 12 h before inoculation and ClNPS6 was strongly expressed at 12-48 h after inoculation.Infection process observation revealed that ClFtr1 affected the production of appressorium 8 hours before infection,but ClNPS6 had no obvious effect on appressorium;after 8 h,ClNPS6 and ClFtr1 did not affect appressorium production.The results of gene expression of key virulence factors in the process of infection showed that the expression of toxin synthesis and cell wall degrading enzyme genes were not significantly different from that of the wild-type strain after ClFtr1 knockout.The expression levels of Clpks18 and Clpmg genes were found after ClNPS6 was knocked out,there was no significant difference in wild-type strains,but the expression levels of Clpg1,Clpg2,Clpg3,and Clpg4 genes decreased significantly.The above results showed that ClFtr1 affected the production of appressorium in the early stage of infection,while ClNPS6 affected the activity of polygalacturonase（PG）in the late stage of infection,regulating the pathogenicity of C.lunata.