The Interaction Mechanism Of Navel Orange-Candidatus Liberibacter Asiaticus-Asian Citrus Psyllids Based On Transcriptomics And Metabolomics

Citrus is one of the most important economic crops in China with the largest cultivated area and production.Asian citrus psyllids（ACP）and Huanglongbing（HLB）seriously restricted the development of citrus industry.HLB was caused by Candidatus Liberibacter asiaticus transmitted by ACP in China.HLB had a lengthy incubation and latent period.Research into citrus-CLas-ACP interactions and the therapy of HLB has been hindered by the lack of persistent pure cultures.It is of positive scientific significance to study the metabolic changes and the defense mechanism of citrus in response to CLas infection and ACP feeding stress.In this study,the metabolic changes of Newhall navel orange leaves infected with HLB in orchards at different stages were analyzed.Healthy ACP（ACP-）and CLas carrying ACP（ACP+）fed on one-year-old navel orange leaves for 24 h and 72 h respectively by bagging method,and the control group（CK）was treated with empty bags to obtain CK,ACP-24 h,ACP+24 h,ACP-72h and ACP+72 h,respectively.Based on metabolomics and transcriptomics,the metabolic regulation mechanism of navel orange leaves in response to ACP-and ACP+feeding stress was comprehensively analyzed.The effects of differential volatiles of navel orange on the behavioral selection of ACP-and ACP+were tested by Y-tube olfactometer,so as to preliminarily understand the interaction mechanism of navel orange-CLas-ACP.The main results were as follows:（1）Matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS）was used to detect significant changes in metabolite and abundance in navel orange leaves in orchards after infection with CLas.Principal component analysis（PCA）showed that leaves with different symptoms of HLB could be clearly distinguished from CK.A total of 32 differential metabolites of navel orange in response to HLB were screened using the projected importance of variables（VIP value）of orthogonal partial least squares discriminant analysis（OPLS-DA）.The contents of citric acid and malic acid decreased with the increase of CLas titer,may be caused by the utilization of host malic and citric acid by CLas for the citric acid cycle（TCA）.MALDI-TOF-MS combined with multivariate analysis established a rapid detection model of HLB from the metabolic level of plant hosts responding to CLas with a discrimination accuracy of 100%,which have the potential for high-throughput and rapid screening of citrus HLB.（2）Volatile organic compounds（VOCs）of navel orange in response to ACP-and ACP+feeding stress were analyzed by dynamic headspace adsorption combined with gas chromatography-mass spectrometry（GC-MS）.A total of 25 VOCs were identified by CK,ACP-24 h,ACP+24 h,ACP-72 h and ACP+72 h,mainly monoterpenes,sesquiterpenes and alcohols,of which 9 were newly added VOCs after stress,including3-carene,sabinene hydrate,cis-linalool oxide,α-terpineol,citronellol,cis-citral,citral,methyl salicylate（Me SA）and neryl acetate.The species and contents of VOCs in Navel orange increased and then decreased with the increase of feeding time,and were most abundant in the ACP+24 h samples.OPLS-DA analysis showed that the differential VOCs between ACP-and ACP+groups for the same treatment time included cosmene,myrcene,3-carene,citral and cis-citral,which may be related to the mediation of CLas.（3）A Y-tube olfactometer was used to test the selection preference of ACP–and ACP+under different concentrations of 8 different VOCs（P<0.05）.It was determined whether each volatile had a significant attracting or avoiding effect on ACP+and ACP-based on the results of selection rate and selection coefficients compared with the air and solvent controls（P<0.05）.The results showed that 6 VOCs had a significant attracting effect on ACP-,including 3-carene,d-limonene,linalool,Me SA,citronellol and citral.While caryophyllene andβ-cyclocitral were attractive to ACP+.However,3-carene,linalool,citronellol and citral had avoidance effects on ACP+.ACP+feeding stress induced the release of metabolites such as 3-carene,d-limonene,linalool,and Me SA in leaves,which in turn attracted ACP-.Volatiles may play an important role in navel orange-CLas-ACP interactions.（4）Non-targeted metabolomics analysis was performed on 5 groups of navel orange leaves based on the ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UPLC-QTOF-MS）technique combined with UNIFI data processing platform.A total of 93 compounds were identified in positive and negative ion modes,including 11 carbohydrates,4 nucleotides,8 amino acids,16 organic acids and 42 flavonoids and their derivatives.ACP-and ACP+significantly reduced the contents of hesperetin,D（+）-glucose,12-Oxo phytodienoic acid,jasmonic acid,and methyl jasmonate,while significantly increased the contents of 5-methylcytidine,eupatilin,turanose,D（+）-glucopyranose 6-phosphate,and 3-p-coumaroylquinic acid.ACP+feeding stress significantly reduced the contents of veronicastroside,eupatorin-5-methyl ether,kaempferide,5,7-dihydroxy-2-（4-hydroxy-3,5-dimethoxyphenyl）-4h-chromen-4-one,sinensetin,jasmonic acid,and 12-oxo phytodienoic acid,while increased the contents of genistein 7-o-glucoside and isocitric acid,possibly due to CLas mediation.Partial least squares discriminant analysis（PLS-DA）showed that there were significant differences in metabolites among 5 groups,which could be well distinguished.The results of OPLS-DA analysis showed that the differential metabolites of ACP-24 h and ACP+24 h included kaempferol-3-O-rutinoside,isorhamnetin 3-O-neohesperoside,citraconic acid,L-malic acid,fumaric acid and isocitric acid,etc.The differential metabolites of ACP-72 h and ACP+72 h included glycerol-3-phosphate,hesperetin,9（S）-Hp OTr E,etc.KEGG pathway analysis showed that metabolites were significantly enriched inα-linolenic acid metabolism,citric acid cycle,flavonoid biosynthesis and other pathways,which may be an important metabolic pathway for navel orange-CLas-ACP interaction.（5）Transcriptomic of 5 group navel orange leaves were studied by next-generation sequencing platform of Illumina.A total of 6188 differentially expressed genes（DEG）were screened by|log₂FC|>1 and P-value<0.05.GO annotation and KEGG enrichment of DEG were conducted.Compared with CK,with the increase of stress time,the number of DEGs induced by ACP-decreased from 3193 at 24 h to 1132 at 72 h,and the number of DEG induced by ACP+decreased from 1873 at 24 h to 879 at 72 h.The number of DEG induced by ACP+was less than that of ACP-,which may be mediated by CLas.The results of GO annotation showed that DEG induced by ACP-and ACP+feeding stress for 24 hours was mainly involved in molecular functions compared with CK,such as ADP binding,heme binding,tetrapyrrole binding,etc.It was mainly enriched in molecular functions and cellular processes after 72 hours of stress,including hydrolase activity,heme binding,tetrapyrrole binding,iron ion binding,chitinase activity,aminoglycan catabolic process,chitin metabolic process,and so on.KEGG pathway significantly enriched in the secondary metabolism and photosynthesis of navel orange,among which phenylpropanoid biosynthesis was the most significant pathway consistent with the results of metabolomic analysis,which might play an important role in the navel orange-CLas-ACP interactions.In addition,a total of 507differential long-stranded non-coding RNA（lnc RNA）were screened from DEG,with the highest expression of LOC102613525,of which 9 were added after feeding stress.Compared with CK,the trends of lnc RNA were different under ACP-and ACP+feeding stress at the same time,which may be related to the mediation of CLas.（6）A total of 129 compounds were identified by GC-MS and UPLC-QTOF-MS analysis.KEGG pathway was significantly enriched in geraniol degradation,salicylic acid metabolism and jasmonic acid metabolism.The results showed that ACP-and ACP+feeding stress induced the release of citral and cis-citral,which was consistent with the expression trend of Cs ADH and Cs ADH3 genes in geraniol degradation pathway.ACP feeding stress induced salicylic acid metabolism of navel orange to release a large amount of Me SA,significantly up-regulated the gene expression levels of some nonexpressor of pathogenesis-related gene（Cs NPR）and pathogenesis-related protein（Cs PR）in systemic acquired resistance（SAR）.The transcription factor that may be involved in the defense response of navel orange was screened as Cs WRKY48.While the gene expression of Cs AOS,Cs AOC,Cs JAR1 and Cs JAZ in jasmonic acid metabolism was down-regulated,which were antagonistic to the salicylic acid metabolism,consistent with the decreasing trend in the levels of compounds related to the jasmonic acid metabolic pathway detected by UPLC-QTOF-MS,including jasmonic acid,9（S）-Hp OTr E,12-Oxo phytodienoic acid,and methyl jasmonate.In this paper,we revealed the metabolomics and transcriptomics change of navel orange responded to ACP-and ACP+feeding stress after 24 h and 72 h.The results showed that ACP-and ACP+feeding induced differences in the metabolism of navel orange VOCs,where ACP+significantly induced the release of 3-carene,d-limonene,linalool and Me SA to attract ACP-,which may be the host regulation mechanism of CLas.ACP feeding stress induced the accumulation of Me SA in salicylic acid metabolism and activated SAR,and then up-regulated the expression of defense genes of Cs NPR,Cs PR,and Cs WRKY48.Salicylic acid signaling were antagonistic to jasmonic acid biosynthesis and inhibited the gene expression ofα-linolenic acid metabolism in navel orange.ACP+feeding stress inhibited the number of differential genes and reduced the content of flavonoids such as veronicastroside.Results showed that CLas mediated the response of navel orange to ACP.The study provided a theoretical basis for the mechanism of citrus-CLas-ACP interactions and contributed to the management of ACP and HLB prevention and control.