Study On The Expression And Regulation Of Citrus Callose Synthase Gene CsCalS5 Under Huanglongbing Stress

Huanglongbing(HLB)is a destructive disease in worldwide citrus,which is mainly caused by a non-cultured,phloem-restricted alpha-proteobacterium,Candidatus Liberibacter asiaticus(CLas)is the main pathogenic species of citrus.The main symptoms of HLB include uneven yellowing of leaves,small or uneven fruit coloration,decline of tree vigor,and even tree death.The HLB characteristics are associated with the callose unnormal deposition in citrus phloem and the obstruction of phytosynthate transportation from source to sink.Callose synthase(CalS)is a key enzyme catalyzing the synthesis of callose,which can be induced in both biotic and abiotic stress.We had found that the expression of CsCalS5 was significantly up-regulated in CLas-infected Citrus reticulata and C.grandis planted in the field.In order to explore the function of callose synthase in the process of citrus-and-CLas interaction,the gene and promoter of CsCalS5 was further studied with the HLB-susceptible variety C.sinensis cv.Jincheng.The coding sequence of CsCalS5 was acquired by PCR from Jincheng genome and was analyzed with bioinformatics softwares.Its relative expression was detected by q PCR in different tissues and under treatment with exogenous phytohormones.The relationship between CsCalS5 expression and callose deposition was revealed in citrus affected with HLB.CsCalS5 P promoter sequence was also cloned and the cis-acting elements were analyzed.PCR and GUS staining were conducted to analyze the marker gene expression in stems and leaf midribs of CsCalS5P::GUS transformed citrus and under exogenous abscisic acid(ABA)and wound stress.The yeast one-hybrid system and dual-luciferase experiment in tobacco were used to screen and verify the candidate protein binding to CsCalS5 P sequence.The research results are as follows:1.Cloning and expression analysis of CsCalS5 gene1)Gene cloning and biological information analysisAccording to the sequence of the CsCalS5 gene in the orange genome,the coding region of the CsCalS5 gene was successfully obtained through cloning and plicing overlap of 4 segments.The sequence was analyzed,we found that CsCalS5 is located on chromosome 7 and is composed of 42 exons and 41 introns.Its open reading frame is5 859 bp and encodes 1 952 aa.The predicted molecular weight of CsCalS5 encoded protein is 224.84 k D,containing 15 transmembrane domains and 3 functional domains,which are Vta1,FKS1 and β-1,3-glucan synthase domains,respectively.2)Expression analyzing of CsCalS5 in different tissueThe expression of CsCalS5 gene was analyzed in different tissues such as the roots,stems and leaves of Jincheng in different developmental stages.The expression of CsCalS5 in the stems was the highest,which was 5.87 times and 5.20.times than that in roots and leaves.In the same tissues with different maturities,the expression level of CsCalS5 in mature tissues was significantly higher than that in young tissues.For example,the expression level of CsCalS5 in 12-month-old leaves was 2.02 times that in1-month-old leaves,and the expression of CsCalS5 in mature stem bark was 16.92 times than that in young stems bark.3)Expression analysis of CsCalS5 induced by phytohormonesCsCalS5 had different responses to exogenous phytohormones such as salicylic acid(SA),methyl jasmonate(Me JA),ethephon(ETH),and ABA.The CsCalS5 gene was significantly up-regulated 3.01 times and 6.60 times after 24 h and 48 h of ABA treatment,comparing with the water treatment group.However,CsCalS5 was not significantly induced or inhibited by exogenous SA,Me JA and ETH.4)Expression analysis of CsCalS5 induced by CLasThe expression of CsCalS5 and the dynamic change of callose deposition were constantly measured.After 7-15 weeks of infection,the expression of CsCalS5 in CLasinfected plants was slightly down-regulated,and the amount of callose deposited was not significantly different comparing with that of healthy controls.The expression of CsCalS5 in CLas-infected plants was up-regulated during 19-47 weeks,and was 4.11,3.04,and 4.53 times that of healthy controls at 23,27,and 39 weeks,respectively.At the same period,the amount of callose deposits increased significantly.The amount of callose deposited in the veins reached 3.25 times that of the control at 39 th week.The results revealed that the expression of CsCalS5 and the amount of callose deposition had the same change trend.2.Cloning and bioinformatics analysis of CsCalS5 P promoter1)Analysis of CsCalS5 P cis-acting elementsThe promoter region(CsCalS5P),1 500 bp upstream of the ATG of the CsCalS5 gene,was cloned and its cis-acting elements were analyzed.The CsCalS5 P sequence contained biotic stress-related elements(GT1 box,dof box and W box),abiotic stressrelated elements(WUN-motif,MYC,ARE,and MBS),and ABA-responsed elenments(ABRE),SA-responsed TCA element,and gibberellin(GA)-responsed GARE motif.2)Creation and tissue-specific expression analysis of CsCalS5P::GUS transgenic plantsThe 35 S promoter of the GNGM1300 vector was replaced with CsCalS5 P to construct a CsCalS5P::GUS fusion expression vector.Five genetic transformation strains were obtained through agrobacterium transformation.Through GUS staining,we found that GUS drove by CsCalS5 P showed high tissue-specific activity in the phloem and cortex of the stems and leaf midribs from the transgenic plants.3)Induced expression of CsCalS5P::GUS transgenic plantsThe leaf discs of the CsCalS5P::GUS transformed strains were treated with ABA,and it was found that the relative expression of the GUS gene was up-regulated in all of5 strains.Among them,the GUS expression in the G23,G29 and G57 strains was 2.09,2.30 and 1.93 times of that of the water control.After 24 hours of wound,the expression of GUS gene in the 5 transgenic lines was up-regulated,and the expression levels of G23,G30 and G58 were 2.10,1.72 and 2.19 times that of 0 h,respectively.3.Screening of candidate factors combined with CsCalS5 P sequenceTwenty five candidate proteins binding to the CsCalS5 P promoter were obtained through a yeast one-hybrid system.ABI5(Cs6g14970),GTE3(Cs8g03030),HMP(Cs9g19170)and Lea5(Cs9g04210)were further confirmed to bind to the CsCalS5 P promoter and activate its transcriptional activity through point-to-point verification in yeast and dual luciferase experiments in tobacco.Among them,ABI5(Cs6g14970)was induced by ABA and CLas,and specificly binds to the CsCalS5 promoter sequence containing ABRE elements.4.Preliminary analysis of CsCalS5-interfered plantThe CsCalS5 gene interference vector and overexpression vector were successfully constructed.Through agrobacterium-mediated genetic transformation,4 interfering strains were obtained.The expression of CsCalS5 in R33,R38,R40,and R41 interference plants was down-regulated by 56.10%,85.30%,83.20% and 46.86%,respectively.The phenotype,microstructure of leaf midribs,and phloem callose deposition points of the interfering strains were not significantly different from those of non-transgenic plants.In summary,citrus callose synthase gene CsCalS5 was highly expressed in phloem and cortical tissues,and regulated by ABA and transcription factor ABI5(Cs6g14970).The expression of CsCalS5 was significantly positively correlated with the deposition of callose in the phloem of citrus under Huanglongbing stress.