Ectopically Expressing Prophage Endolysin Genes From Candidatus Liberibacter Asiaticus Confer Resistance To Huanglonbing In Citrus

Citrus Huanglongbing(HLB),caused by Candidatus Liberibacter asiaticus(Clas)is the most devastating disease in citrus worldwide.At present,HLB has occurred in more than 50 countries in the world,which has caused serious economic losses to citrus industry in many countries including China.So far,no method has been developed to cure the HLB infected citrus trees,and no germplasm and cultivar in Citrus possessed resist to the HLB.Introduction of resistance from other genetic resources into citrus variety is the most fundamental and effective way to control HLB disease.In this study,two prophage endolysin genes CaLasLYS1 and CaLasLYS2 were derived from the genome of citrus Huanglongbing baterium.The purified proteins were obtained by prokaryotic expression of the two genes.The antibacterial activity of the prokaryotic expressed proteins was evaluated by antibacterial experiments.The plant expression vectors of CaLasLYS1 and CaLasLYS2 genes were constructed by optimizing the plant preference codons of two genes.The two genes were introduced into the genome of citrus rootstock Carrizo citrange(Citrus sinensis Osbeck.× Poncirus trifoliata Raf)by Agrobacterium-mediated method.After GUS positive bud screening,PCR and Western blotting detection,transgenic citrange plants with high expression of the target gene were obtained.The transgenic plants were infected with Huanglongbing pathogen by leaf disc grafting.Through the evaluation of HLB resistance,the transgenic lines resistant to Huanglongbing were successfully selected.The DTBIA method was used to detect the distribution of CLas in the aboveground and underground parts of transgenic plants.The effects of transgenic CaLasLYS1 and CaLasLYS2 genes on the proliferation of CLas,endophytic bacterial diversity and immune defense response in citrus plants were investigated by root microbial community structure and comparative transcriptome analysis.The main research results achieved in this study are as follows:1.Bioinformatics analysis of CaLasLYS1 and CaLasLYS2 genes and charactiertics of their expression in HLB infected citrange and psyllaTwo citrus CLas prophage endolysin-related genes,CLIBASIA04790 and CLIBASIA04800,were obtained from the genome of citrus CLas(psy62),which were named CaLasLYS1 and CaLasLYS2 in this study,respectively.The proteins CaLasLYS1 and CaLasLYS2 encoded by these two genes were 100 % matched with the amino acid sequences of lysozyme C6XGM6 and C6XGM8,respectively.Both C6XGM6 and C6XGM8 belong to the glycoside hydrolase 24 subfamily(GH24),which can hydrolyze the β-1,4 glycosidic bond of N-Acetylmuramic Acid and N-Acetylglucosamine residue in peptidoglycan to cleave the cell wall of bacteria.The 3’-terminal amino acid sequence of CaLasLYS1 has 77.4 % homology with the sequence of CaLasLYS2,of which 48 amino acid sequences are identical.However,CaLasLYS1 has 70 more amino acid residues at the N-terminus than CaLasLYS2,including three catalytic residues Glu40,Asp49 and Thr56.CaLasLYS1 contains seven H1 – H7α-helices and four S1 – S4 β-sheets,while CaLasLYS2 contains six H2 – H7 α-helices and two S3 and S4 β-sheets.In addition,CaLasLYS1 and CaLasLYS2 are single-domain globular proteins,in which CaLasLYS1 contains 3D domain 1 and domain 2,including H1 α-helix,S1 and S2 β-sheet,H2 to H7 α-helix,and S3 and S4β-sheet,respectively.CaLasLYS2 contains only domain 2.The expression of CaLasLYS1 and CaLasLYS2 genes in the psyllids carrying CLas was significantly higher than that in the HLB infected citrus plants.The expression of CaLasLYS1 and CaLasLYS2 genes in leaves was significantly higher than that in roots,and the expression in non-symptomatic leaves was significantly higher than that in symptomatic leaves.2.Bacteriostatic activities of CaLasLYS1 and CaLasLYS2More than 3mg of proteins with a purity of 80 % of CaLasLYS1 and CaLasLYS2 were obtained by prokaryotic expression of CaLasLYS1 and CaLasLYS2 genes,of which CaLasLYS2 protein was an inclusion body.The results of Oxford cup method showed that CaLasLYS1 and CaLasLYS2 had obvious antibacterial effect on Gram-negative bacteria EHA105,but had no antibacterial effect on Gram-positive bacteria HD73.Further antibacterial tests were performed on different strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes,Escherichia coli DH5α,citrus canker Xcc and other Gram-negative bacteria.It was found that CaLasLYS2 protein had obvious inhibition zones on A.tumefaciens LBA4404,EHA101,GV3101,A.rhizogenes MSU440,Ar1193,and the diameter of the inhibition zone was between0.823 ± 0011 cm – 1.253 ± 0.044 cm.It also had certain antibacterial effect on Xcc,and the diameter of the inhibition zone was 0.786 ± 0.006 cm.However,CaLasLYS1 protein did not form an inhibition zone against the above six strains,indicating that the antibacterial effect of CaLasLYS1 and CaLasLYS2 proteins was selective,and CaLasLYS2 protein had a broad antibacterial spectrum.3.Introduction of CaLasLYS1 and CaLasLYS2 into citrangeThe plant preference codons of CaLasLYS1 and CaLasLYS2 genes were optimized,and the plant expression vectors of the target genes controlled by the 35 S promoter were constructed.The optimized CaLasLYS1 and CaLasLYS2 genes were introduced into the genome of Carrizo citrange by A.tumefaciens-mediated method.Numbers of GUS positive regenerated plants were generated.After PCR identification,18 CaLasLYS1 transgenic plants and 9 CaLasLYS2 transgenic plants were selected.Real-time quantitative PCR(q RT-PCR)analysis showed that the target gene was highly expressed in all transgenic lines.The expression levels of the target gene proteins in the leaves and roots of the transgenic plants was further detected by semi-quantitative Western blotting.The protein expressed by the target gene was detected in the transgenic plants,and the protein expression level of each line was equivalent to the protein level of the internal reference gene actin,and no significant difference between the transgenic lines.4.Expressions of CaLasLYS1 and CaLasLYS2 in citrange generated the HLB resistanceThe transgenic self-rooted citrange plants with high expression level of the target gene and consistent growth were selected and inoculated with HLB by leaf disc grafting.After 20 months of inoculation,the HLB disease rate of wild-type(WT)plants was75 %,the HLB disease rate of CaLasLYS1 transgenic plants was 66.7 %,and the HLB disease rate of CaLasLYS2 transgenic plants was 25 %.Some of the HLB diseased plants of the WT showed HLB symptoms such as root rot,leaf curl,and plant dwarf after 4 months of inoculation.After 20 months of inoculation,a small number of CaLasLYS1 transgenic plants showed severe symptoms.Most of the CaLasLYS2 transgenic plants had no obvious symptoms,and the plants grew normally.Some transgenic lines were only detected HLB pathogens in the first 3 months after inoculation,and then no HLB pathogens were detected in those plants.Through the comprehensive evaluation with the above three aspects of resistance evaluation,three CaLasLYS1 transgenic plants and one CaLasLYS2 transgenic plant with HLB tolerance,and two CaLasLYS2 transgenic plants with high HLB resistance were screened.DTBIA detection showed that there was a high concentration of CLas in the phloems of roots,stems and leaves of CaLasLYS1 transgenic and WT plants with Huanglongbing tolerance,while there was almost no CLas in the roots,stems and leaves of CaLasLYS2 transgenic plants with high resistance to Huanglongbing.This result further confirmed that the CaLasLYS2 transgenic plants had resistance to Huanglongbing.5.Profiles of microbial community of transgenic citrangesTransgenic lines of CaLasLYS1-2 and CaLasLYS2-6 with resistance to Huanglongbing were selected to analyze the diversity of endophytic microorganisms in roots and leaves of transgenic plants.The results showed that the endophytic bacterial communities in roots of CaLasLYS2-6 and WT were different after inoculation of CLas,while the fungal communities in roots were only different before inoculation.The abundance of endophytic bacterial species and fungal communities in leaves of CaLasLYS1-2 and WT were different before inoculation,and the abundance of endophytic bacterial communities and fungal species in roots were different after inoculation.It indicated that the microbial community in the leaves or roots of CaLasLYS2-6 and CaLasLYS1-2 had different interaction patterns.After inoculation,the abundance of rhizobia,Sphingomonas and Burkholderia in the roots of CaLasLYS2-6 was significantly higher than that of WT.Before inoculation,the abundance of arbuscular mycorrhizal fungi Heterorhizobium sp.in the roots of CaLasLYS2-6 was significantly higher than that in WT,while the abundance of Neochaeta sp.was significantly lower than that in WT.The change of microbial community structure in resistant transgenic plants indicated that the prophage endolysin of HLB also had selective bacteriostatic effect on citrus endophytic bacteria,and the resulting microbial community may induce different host defense responses and affect the resistance of HLB.6.Transcriptome analysis of transgenic citrangeThe changes of endophytic bacteria can often change the immune response of the host.In order to explore the effect of the changes of endophytic bacteria in CaLasLYS1 and CaLasLYS2 transgenic plants on the host defense response,the transcriptome sequencing analysis of CaLasLYS1-2 and CaLasLYS2-6 lines was carried out with WT plants as the control.The analysis results showed that the systemic immune resistance of CaLasLYS1 and CaLasLYS2 transgenic plants was enhanced,and the expression level of innate immune resistance genes was changed.GO functional enrichment analysis showed that the differentially expressed genes were mainly related to the metablic process,cellular process,single-organism process,biological regulation,response to stimulus,and localization.Cellular component organization or biogenesis,signaling,etc.KEGG metabolic pathway analysis showed that the differentially expressed genes were significantly enriched in plant hormone signal transduction(ko04075),photosynthesis-antenna protein(ko00196),photosynthesis(ko00195),circadian rhythm-plant(ko04712),plant-pathogen interaction(ko04626)pathways etc.These genes mainly include 6 transcription factors,PTI1-4,WRKY18,WRKY40,WRKY53,b HLH(Upa20)and ERF1,4 disease resistance proteins RPM1 and RPS2,2 respiratory burst oxidase homologue proteins RBOHB and RBOHD,and 17 kinase family protein genes,of which WRKY40,WRKY18,MEKK1 and RBOHD may play a key role in the resistance of transgenic plants.The above results showed that CaLasLYS1 and CaLasLYS2 had specific antibacterial effect on bacteria.The two prokaryotic expressed proteins had antibacterial effect on some Gram-negative bacteria,but had no antibacterial effect on the tested Gram-positive bacteria,and the antibacterial spectrum of CaLasLYS2 was significantly broader than that of CaLasLYS1.Expressions of CaLasLYS1 and CaLasLYS2 citrange by transgenic technology produced resistance to Huanglongbing.The CaLasLYS2 transgenic lines showed stronger resistance to Huanglongbing than the CaLasLYS1 transgenic lines.The root microbiome composition of CaLasLYS1 and CaLasLYS2 transgenic lines was changed,and the abundance of beneficial bacteria was significantly increased.The expression levels of genes related to innate immune resistance in CaLasLYS1 and CaLasLYS2 transgenic plants were significantly up-regulated,and systemic immune resistance was enhanced.