Regulation Of Serine Palmitoyl Transferases StLCB1 And StLCB2 On Pathogenicity Of Setosphaeria Turcica

Northern Corn Leaf Blight is one of the most important leaf blight in corn,which often causes serious yield reduction and leads to great economic losses.In the pathogenic process,Setosphaeria turcica mainly adhered to the host leaf epidermis by conidia,and the end of the germ tube expanded to form appressorium after germination.The appressorium accumulates turgor pressure and secretes cell wall degradation enzymes,and finally forms invasion nails,which penetrate the host epidermis under mechanical pressure and complete the infection process.Sphingolipids are essential components of eukaryotic cells.Their metabolites give membranes important structural properties and are regulatory molecules in a variety of cellular processes.Sphingosine is the most important intermediate product in sphingolipid metabolism pathway.Serine palmitoyl transferase catalyzed the de novo synthesis of sphingosine and was the rate-limiting enzyme in this pathway.Our previous research found that treatment of Myriocin,a specific inhibitor of serine palmitoyl transferase,could cause abnormal development of appressorium in S.turcica.In this study,metabolite data were obtained during the appressorium formation of S.turcica.The data indicated that sphingosine content significantly increased during the appressorium stage of S.turcica.Therefore,we used SKI Ⅱ,a specific inhibitor of sphingosine kinase,to increase the accumulation of sphingosine,and created the mutants of LCB1 and LCB2,which encoded the catalytic subunit of SPT to study the relationship between sphingoid and pathogenicity regulation of S.turcica.The main research results were as follows:1.The LCB1 coding gene with ID 167454 was searched in the genomic data of S.turcica published by JGI and named StLCB1.The total length of the gene was 3987bp.Its CDS was 1527 bp,encoding 509 amino acids.The LCB2 coding gene with ID 73160 was named as StLCB2.The total length of the gene was 3974bp with a 1974bp CDS sequence encoding 658 amino acids.2.The result of sphingosine kinase inhibitor(SKI Ⅱ)treating conidia of S.turcica showed that at the 6th hour of conidial development,the formation rate of appressorium increased with the increase of inhibitor concentration,but too high concentration inhibited the development of appressorium.The appressorium formation ratio with 500 μmol/L SKI II was 93.2%,which was higher than that of control group(75.5%).In the pathogenicity test,the number of early infection points in the experimental group was 353,which was higher than that in the control group(182).These results indicated that the balance between sphingosine and sphinganine-1-phosphate plays an important role in the development of conidial infection structure.3.The StLCB1 gene over expression vector and StLCB2 gene silencing vector were transformed into the wild-type 01-23 of S.turcica by PEG-mediated protoplast transformation.In this study,two 3HA::eGFP::StLCB1 over-expression mutants named pBARKS1-StLCB1-1 and pBARKS1-StLCB1-2 were obtained,screened by hygromycin B resistance,PCR,qRT-PCR and Western blot.Three silent mutants named RNI-StLCB2-1、RNI-StLCB2-30 and RNI-StLCB2-49 were obtained,screened by hygromycin B resistance,PCR,Semi-quantitative RT-PCR,qRT-PCR.The expression levels of StLCB1 gene in WT and mutants were analyzed by qRT-PCR.The results showed that the expression level of StLCB1 gene in two overexpression mutants was significantly higher than that in WT,which was 3.1 and 4.3 times of that in WT,respectively.In three silent mutants,the expression level of StLCB2 gene was lower than that of WT,which was 0.51,0.35 and 0.47 times of that of WT,respectively.4.Compared with WT strain,StLCB1 overexpressed mutant had dense airborne mycelium,dark brown colony,and intracellular melanin content was 56.21 mg/g,which was 1.39 times as much as that of WT strain.Extracellular melanin content was 6.94 mg/g,1.34 times as much as that of WT,and the average hyphal septa spacing was 1.71 times higher than that of WT.The airborne mycelium of StLCB2 silent mutant was slender and dense,felt-like,the colony was gray and white,and the intracellular melanin content was 7.3 mg/g,which was 0.18 times as much as that of WT.Extracellular melanin content was 1.84 mg/g,which was about 0.35 times as much as that of WT.Almost no conidia was produced,and the average spacing between hyphal septa was 0.28μm,which was about 4 times as long as that of WT,and the growth rate slowed down.These results indicated that StLCB1 and StLCB2 genes could regulate mycelial growth and conidium development of S.turcica.5.The formation and penetration of appressorium were observed by cellophane induction,and the pathogenicity of appressorium was detected by infected host.The results showed that the appressorium formation rate of StLCB1 overexpressed mutant was 51.3%at 24h induction,which was higher than that of WT(30.2%).At 48h of induction,the penetration rate of appressorium was 30.1%,lower than 66.4%of WT.The terminal enlargement rate of StLCB2 RNAi mutant was only 3.86%at 24h induction,and its penetration rate was 6.72%at 48h after induction,which was much lower than that of WT.On the susceptible host B73 inbred line,the number of early infection points of maize leaves inoculated with StLCB1 mutant and StLCB2 silent mutant was 137 and 37,respectively,which were both lower than 253 infection points of the leaves inoculated with WT.After 12 days of inoculation,yellow brown necrotic lesions appeared on maize leaves,and the incidence area of mutant leaves was small,especially StLCB2 silent mutant.These results suggest that StLCB1 and StLCB2 genes play important roles in regulating appressorium development and pathogenicity.6.The sphingosine content was determined in mutants and WT.The results showed that the sphingosine content of StLCB1 mutant was slightly higher than that of WT,but not significantly.The sphingosine content of StLCB2 silent mutant decreased to 0.79 times of that of WT.In the StLCB1 overexpression mutant,the expression of StLCB2 gene did not change significantly,but in the StLCB2 silencing mutant,the expression of StLCB1 gene decreased to 0.65 times of that of WT.This indicates that StLCB1 and StLCB2 do not have functional redundancy,but interact with each other to synergistically regulate sphingosine synthesis.7.The membrane integrity of mutant cells was analyzed by PI(Propidium iodide)staining.The results showed that under fluorescence microscope,only the broken ends of mycelia showed red fluorescence in WT and StLCBl overexpression mutants,and the nuclei showed almost no staining phenomenon,indicating that the cell membrane of StLCB1 overexpression mutants remained intact,while StLCB2 silent mutant cells almost all showed red fluorescence,indicating that StLCB2 silencing caused damage to the integrity of cell membrane.Based on the control mechanism of morphogenesis of infection structure,this study conducted in-depth research on the role of Sphingosine in pathogen infection,and clarified the regulation of sphingosine on the formation of appressorium,the polar growth of hyphae,the integrity of cell membrane,and the pathogenicity of corn leaf spot pathogen,laying a foundation for exploring new disease prevention and control strategies.