| Citrus Huanglongbing（HLB）is a devastating disease in citrus,which has caused huge economic loss to the world citrus industry.The disease is mainly caused by Candidatus Liberibacter asiaticus（CaLas）,which is difficult to culture in vitro.At present,no effective cures and no disease resistant varieties are available in citrus industry.Breeding HLB-resistant varieties is the fundamental way to address this disease.Different citrus varieties and citrus related varieties have obvious differences in resistance to HLB.However,the mechanism of HLB resistance in citrus is still unclear,and its key regulatory genes and molecular mechanisms need to be further understood.Jincheng orange（Citrus sinensis Osbeck,JC）is susceptible to HLB,and kaffir lime（Citrus hystrix,KL）is tolerant to HLB.Orange jasmine（Murraya paniculata（L.）Jack,OJ）,belonging to Rutaceae,is a favorite plant of citrus psyllid,and has no obvious symptoms after being infected by CaLas.OJ is considered to be an HLB-resistant species,but its disease-resistance mechanism is still unclear.In this study,we characterized the response profilings of the three varieties to HLB by grafting inoculation using JC branches with CaLas as scions.The resistance characteristics of OJ,KL and JC to HLB were determined based on the severity of plant symptoms and the proliferation of pathogens.Further,the transcriptome differences of OJ,KL and JC in response to CaLas were compared using RNA-seq technology.Combined with the change characteristics of plant hormones,phloem tissue structure,callose deposition and starch accumulation induced by CaLas,the important genes,functions and pathways in response to HLB were identified.To provide theoretical reference for citrus molecular breeding of resistance to HLB,the possible resistance mechanisms of citrus were discussed based on the above results.The main results are as follows:1.Comparative analysis of OJ,KL and JC resistance to HLBSymptoms Characteristics.In six months after Las infection,JC showed obvious symptoms including mottled yellowing of leaves and lignification of leaf veins.Comparatively,KL showed slight yellowing symptoms in a few leaves,but had severe lignified burst in leaf veins.OJ has no visible symptoms.During the observation of 18months,the disease of JC,accompanied by severe defoliation,was more serious than that of KL;OJ never showed any symptoms.Growth characteristics of CaLas.The continuous detection of CaLas content for six months showed that the proliferation rate of CaLas in JC was the fastest and always maintained an increasing trend.In KL,CaLas proliferated rapidly in the initial stage,but slowed down in the later stage.The contents of CaLas in OJ were very low and the proliferation rate was also very slow.Comparatively,the contents of CaLas in OJ were significantly lower than these in JC and KL,and averagely,it was lower by 300 and 20times compared to JC and KL,respectively.2.Comparative analysis of plant hormone in response to CaLas in OJ,KL and JCCaLas infection significantly affected the accumulation of SA and Me SA,JA,GA,Br,BA and IAA in the three varieties.Before CaLas infection,the content of SA in OJ was the highest,followed by KL,and its content in JC was the lowest.After CaLas infection,the SA content in OJ and KL decreased significantly,but it increased significantly in JC.The data suggests that the basic level of SA is closely related to the resistance of citrus varieties,and the accumulation of SA induced by CaLas seems to be detrimental to citrus resistance.Before CaLas infection,the JA content of JC was significantly higher than that of KL and OJ.After CaLas infection,the JA content of the three varieties decreased significantly,implying CaLas negatively regulates the accumulation of JA in citrus.After CaLas infection,the contents of IAA and GA increased significantly in JC and KL,but decreased significantly in OJ.3.Summary of transcriptomes of OJ,KL and JC in response to CaLasAfter four months of infection with CaLas,the same mature leaves were selected for transcriptome sequencing analysis.The healthy leaves of three varieties were used as controls,and three biological repeats were set.Compared to healthy controls,OJ,KL and JC had 1,954,1,549 and 1,065 differentially expressed genes（DEGs）,respectively.In JC,772 and 293 DEGs were up-regulated and down-regulated by CaLas,respectively.In KL,1,249 and 300 DEGs had increased and decreased experession in response to CaLas.Both the number of DEGs up-regulated and down-regulated by CaLas was 977 in OJ.Cluster analysis showed that the transcriptional abundances of 2,009 genes were similar in three varieties.However,the transcription levels of 1,859 genes in JC and KL were significantly higher than that in OJ,indicating that the CaLas induced an overall increase in the transcriptional activities of JC and KL,but has little effect on that of OJ.4.Transcriptional characteristics of biostress-related pathways in response to CaLasMapman comparative analysis revealed that CaLas significantly up-regulated biostress-related pathways in OJ.Compared with JC,the transcriptional activities of NLRs resistance protein genes,which were related to pathogen recognition and host resistance,and to bacterial flagellin receptors FLS2,Ca2+and ROS mediated immune pathways were significantly affected by CaLas in OJ and KL.The results indicated that the ability of host innate immune response to CaLas infection played a key role in Citrus Huanglong resistance.5.Response characteristics of the phloem in response to CaLasMicroscopic observation uncovered that the phloem cell layers increased,the cell wall thickened,and the cell arrangement was disordered in JC and KL after CaLas infection.The proliferation rate of the phloemic cells in KL was faster than that of JC.Compared with the healthy control,the number of phloem cell layers of OJ did not change significantly,but its phloem tissue was significantly thickened.The results showed that the excessive proliferation of phloem in response to CaLas infection was not conducive to HLB-resistance of citrus.Transcriptome analysis discovered that CLE41,ethylene,auxin and cytokinin mediated signaling pathways were involved in the regulation of phloem proliferation in response to CaLas infection in citrus.The transcriptional activities of regulatory genes,related to phloem development（such as PXY,WOX14,and TMO6）were significantly stronger in JC and KL than that in OJ,which was consistent with the proliferation of phloem cells in JC and KL.6.Response characteristics of the starch metabolism to CaLasThe determination of starch content in leaves showed that in healthy plants,the starch content of KL was much higher than that of JC and OJ plants.Compared with healthy controls,the starch contents in KL and JC decreased significantly by 1.4 and2.7 times while it decreased slightly in OJ.In OJ,KL and JC infected by CaLas,7,8 and 12 DEGs were annotated to sucrose-starch metabolism,respectively.The expression levels of DEGs,annotated to starch synthesis pathway,was significantly higher in JC and KL than that in OJ,showing that the starch synthesis in JC and KL was stronger than that in OJ during CaLas infection.7.Response characteristics of callose in response to CaLasThere was no significant difference in callose deposition among healthy OJ,KL and JC.Callose deposition in JC and KL was very serious after CaLas infection and it increased by 6 times and 13 times in the two varieites,respectively,compared with healthy controls.Compared with healthy control,CaLas-infected OJ had a slight decrease in callose deposition,but there was no significant difference.In JC and KL infected by CaLas,the transcriptional activities of genes,encoding callose synthesis complexs,were much higher than that of OJ.The PP2（Phloem Protein 2）genes involved in callose deposition in phloem were up-regulated by CaLas.Among them,six PP2 DEGs were detected in KL,but only one in OJ and JC.The results showed that the callose synthesis and deposition of JC and KL were significantly more active than that of OJ during CaLas infection. |
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