Development Of Transgenic HLB Resistance In Citrus With NPR And Antimicrobial Genes

Citrus Huanglongbing(HLB),mainly caused by Candidatus Liberibacter asiaticus(CLas),is the most devastating bacterial disease in citrus production,which has caused enormous economic losses to citrus industry worldwide.Currently,the resistance to HLB has not been found in the Citrus,also no any approach could thoroughly eliminate the CLas from infected citrus plants.Breeding HLB-resistant citrus varieties is the most fundamental method to control the disease,which is very important for maintaining the sustainable and healthy development of citrus industry.At present,there are two strategies applied for HLB-resistant breeding.One is to induce plants to generate systemic acquired resistance,the other is the expression of exogenous genes which can kill pathogens in plants.Therefore,in this study,based on the above-mentioned two anti-disease strategies,two nonexpressor of pathogenesis-related(NPR)genes Ciclev10031343 m and Ciclev10031749 m,which are involved in systemic acquired resistance,were selected from HLB-tolerant (?)Jackson(?) grapefruit(Citrus paradisi Macf.)and termed as CiNPR3 and CiNPR4,respectively,by homologous genes and phylogenetic tree analysis.Meanwhile,two fusion genes HPC and HAC were synthesized by connecting human lysozyme and antibacterial peptide Cecropin B genes with connecting polypeptides LP4 and 2A,respectively.CiNPR3,CiNPR4,HPC and HAC were introuduced into plant overexpression vector,respectively.Transgenic plants were produced by introducing CiNPR3,CiNPR4,HPC and HAC,respectively,into the genome of HLB-susceptible (?)Wanjincheng(?) orange(C.sinensis Osbeck).The stable expression of target gene was confirmed by quantitative real-time PCR.CLas was inoculated to transgenic plants by grafting CLas-infected bud.Resistance of transgenic plants to HLB was evaluated in a greenhouse at 28? for 2 years after inoculation by investigating the CLas population and incidence rate.Then,the starch content and phloem structure of HLB-resistant transgenic plants were analyzed.The mechanisms of CiNPR3 and CiNPR4 regulating resistance to HLB were studied by comparative transcriptomic analysis,interacting protein analysis,and the expression analysis of the genes related to defense response in the signaling pathway mediated by salicylic acid(SA)and jasmonic acid(JA).The effect of HPC or HAC on the development of HLB resistance in citrus was evaluated by analyzing the expression level of callose synthase gene and starch synthesis-related genes in HPC and HAC transgenic plants.The main research results are as follows:1.Production of transgenic plants with overexpressed CiNPR3,CiNPR4,HPC or HACCiNPR3,CiNPR4,HPC and HAC genes were introduced into HLB-susceptible (?)Wanjincheng(?) orange,respectively,by Agrobacteria-mediated genetic transformation,in order to study their effects on HLB resistance.Thirty CiNPR3,26 CiNPR4,56 HPC and 64 HAC transgenic lines were obtained.The expression of CiNPR3 and CiNPR4 in CiNPR3 and CiNPR4 transgenic plants,respectively,both human lysozyme and Cecropin B genes in HPC and HAC transgenic plants was confirmed by quantitative real-time PCR.The expression of HPC and HAC fusion protein in HPC and HAC transgenic plants,respectively,was confirmed by Western blotting analysis.The morphological phenotype of transgenic plants was not different from that of wild-type(WT)plants.2.Overexpression of CiNPR3,CiNPR4,HPC or HAC in transgenic citrus plants demonstrated resistance to HLBFrom 9 months after inoculation(MAI)of CLas,CLas population in phloem of transgenic citrus plants was investigated.CLas population in 57.1%(16/28)of CiNPR3 lines,61.5%(16/26)of CiNPR4 lines,72.7%(16/22)of HPC lines and 57.1%(12/21)of HAC lines were significantly lower than that in WT plants.At 18 MAI,10 out of 16 CiNPR3,7 out of 16 CiNPR4,10 out of 16 HPC and 7 out of 12 HAC transgenic lines had significantly lower CLas population than WT plants.At 24 MAI,still 9 CiNPR3,7 CiNPR4,7 HPC and 7 HAC transgenic lines showed significantly lower level of CLas population than WT plants.These results indicated that overexpression of CiNPR3,CiNPR4,HPC or HAC significantly inhibited the proliferation of CLas pathogen.At 9 MAI,obvious HLB symptoms were observed in WT plants and some transgenic plants.The newly sprouted leaves on them turned yellow and became smaller,and some of leaves showed prominent veins.Over time,the plants with HLB symptoms grew slowly and some shoot tips gradually dried.28.6%(8/28)of CiNPR3,23.1%(6/26)of CiNPR4,27.3%(6/22)of HPC and 19.0%(4/21)of HAC transgenic lines did not show obvious HLB symptoms at 24 MAI.The results indicated that overexpression of CiNPR3,CiNPR4,HPC or HAC reduced the CLas population in phloem,therefore,alleviated the HLB symptoms of transgenic plants,and demonstrated certain level of resistance to HLB.3.Changes in the anatomical structure and starch content of transgenic plants in response to CLas infectionAt 24 MAI,phloem structures in HLB-resistant CiNPR3,CiNPR4,HPC and HAC plants without and with CLas-infection were anatomically observed.The phloem structures of non-infected CiNPR3,CiNPR4,HPC and HAC transgenic plants had no difference from that of non-infected WT plants.Compared with non-infected WT plants,the phloem layer of CLas-infected CiNPR3,CiNPR4,HPC and HAC plants became wider,and was filled with enlarged loose parenchyma cells,but regularly distributed.Over accumulation of callose in the phloem was observed in all four kinds of transgenic plants.However,compared with CLas-infected CiNPR3,CiNPR4,HPC and HAC plants,CLas-infected WT plants had much wider phloem layer with larger space among disorderly distributed parenchyma cells,and higher level of accumulation of callose in the phloem.These results indicated that overexpression of CiNPR3,CiNPR4,HPC or HAC did not change the phloem structure of (?)Wanjincheng(?) orange,but alleviated the damage of phloem structure by CLas.The starch content in leaf of HLB-resistant CiNPR3,CiNPR4,HPC and HAC plants were determined at 24 MAI.The starch contents in leaves of all tested transgenic plants increased significantly compared with non-infected WT plants,but were significantly lower than that in CLas-infected WT plants,indicating that overexpression of CiNPR3,CiNPR4,HPC or HAC reduced starch accumulation in leaves of HLB-resistant transgenic citrus plants induced by CLas infection.4.Mechanisms of resistance to HLB by overexpression of CiNPR3 or CiNPR44.1 Analysis of transcriptomic profiles of CiNPR3 or CiNPR4 transgenic plantsAt 18 MAI,the HLB-resistant CiNPR3 and CiNPR4 transgenic plants C3-29 and C4-2,also WT plants,without and with CLas-infection,were selected for transcriptomic analysis.The metabolic or signal transduction pathways involved by differentially expressed genes(DEGs)were analyzed by KEGG enrichment analysis.Before infection,compared with non-infected WT plants,DEGs from CiNPR3 transgenic plant C3-29 were significantly enriched in photosynthesis-antenna proteins,amino sugar and nucleotide sugar metabolism,and photosynthesis,DEGs from CiNPR4 transgenic plant C4-2 were significantly enriched in photosynthesis-antenna proteins,alpha-linolenic acid metabolism and plant hormone signal transduction.After infection,compared with respective non-infected plant,DEGs from CiNPR3 transgenic plant C3-29 were significantly enriched in phenylalanine metabolism and protein processing in endoplasmic reticulum,DEGs from CiNPR4 transgenic plant C4-2 were significantly enriched in plant?pathogen interaction,photosynthesis and alpha-linolenic acid metabolism,however,DEGs from WT plants were significantly enriched in limonene and pinene degradation,stilbenoid,diarylheptanoid and gingerol biosynthesis pathway.These results indicated that overexpression of CiNPR3 or CiNPR4 did not significantly enhance the basic defense response of transgenic plants,but that overexpression of CiNPR4 enhanced the pathogen-induced basic resistance of transgenic plants.4.2 CiNPR3 and CiNPR4 proteins interacted with CsTGA6 and CsTGA2 transcription factors,respectivelyBoth CiNPR3 and CiNPR4 proteins from (?)Jackson(?) grapefruit interacted with the proteins encoded by Ciclev10005080 m and Ciclev10001081 m genes as revealed by analyzing sub-network of protein-protein interaction.The amino acid sequence encoded by Ciclev10005080 m gene is identical to that encoded by Cs5g11160.1 gene,while the amino acid sequence encoded by Ciclev10001081 m is 98.9% similar to that encoded by Cs1g16230.4 gene.Cs1g16230.4 and Cs5g11160.1 proteins belong to TGA6 and TGA2 transcription factors,respectively.So,in this study,Cs1g16230.4 and Cs5g11160.1 were termed as CsTGA6 and CsTGA2,respectively.Yeast two-hybrid analysis confirmed that CiNPR3 and CiNPR4 proteins interacted with CsTGA6 and CsTGA2 transcription factors,respectively.4.3 Overexpression of CiNPR3 or CiNPR4 significantly improved systemic acquired resistance and induced systemic resistance of transgenic plantsAt 18 MAI,the expression level of SA-and JA-mediated the marker genes of defense response CsPR1,CsPR2 and CsPR5,and CsPDF1.2,respectively,in HLB-resistant CiNPR3 and CiNPR4 transgenic plants without and with CLas infection was analyzed.Before infection,compared with non-infected WT plant,the expression level of CsPR1 in all tested CiNPR3 and CiNPR4 transgenic plants was significantly up-regulated,the expression level of CsPR2 was significantly down-regulated in all tested CiNPR3 transgenic plants,and significantly down-regulated or no difference in CiNPR4 transgenic plants,the expression level of CsPR5 in all tested CiNPR3 and CiNPR4 transgenic plants was no significantly difference,and the expression level of CsPDF1.2 was significantly up-regulated or no difference in tested CiNPR3 and CiNPR4 transgenic plants.After infection of CLas,WT plants had up-regulated expression levels of CsPR1 and CsPR2,basically unchanged expression level of CsPR5,and slightly down-regulated expression level of CsPDF1.2.However,the expression levels of CsPR1,CsPR2,CsPR5 and CsPDF1.2 in all tested CiNPR3 and CiNPR4 transgenic plants were significantly higher than those in WT plants.These results indicated that overexpression of CiNPR3 or CiNPR4 not only increased CsPR1-mediated the basal defense response and JA-mediated induced systemic resistance caused by CLas infection,but also enabled transgenic citrus plants to acquire stronger CLas-induced systemic acquired resistance than WT plants.In summary,overexpression of CiNPR3 strongly activates the expression of SA-mediated PR genes and JA-mediated PDF1.2 gene by binding with CsTGA6 transcription factor and initiates the enhanced systemic acquired resistance and induced systemic resistance of transgenic plants to gain the resistance to HLB.In addition to the same working mode as CiNPR3,CiNPR4 enhances the pathogen-induced basic resistance of transgenic plants by activating the transcription of genes involved in plant?pathogene interaction pathway.5.Expression of callose synthase gene and starch synthesis-related genes in HPC and HAC transgenic plants in response to CLas infectionAt 24 MAI,the expression levels of callose synthase gene and starch synthesis-related genes in HLB-resistant HPC and HAC transgenic plants were analyzed.The results showed that the expression level of callose synthase gene Cs Cal S7 in HPC and HAC transgenic plants was significantly up-regulated compared with WT plants,and that the expression levels of starch synthesis-related genes,such as ADP-glucose pyrophosphorylase gene Cs SS13(Cs8g07230.1)and starch synthase gene Cs SS14(Orange1.1t00566),were significantly lower in HPC and HAC transgenic plants than in WT plants.These results indicated that overexpression of HPC and HAC had significant great impact on expression of callose synthase gene Cs Cal S7,starch synthesis-related genes Cs SS13 and Cs SS14 in transgenic plants in response to CLas infection.