Cytopathology And Host Transcriptome Analyses Of Citrus Huanglongbing

Citrus Huanglongbing ([HLB]) and Tristeza are two of the most destructive citrus diseases which are becoming more serious in recent years than ever before throughout the world. HLB has been found in approximate50countries since its first report in1913in China, and Tristeza is even much widely distributed. HLB has been causing great reduction of citrus production in Florida, USA since2004and in Sao Paulo, Brazil since2005, respectively. HLB is associated with three species of phloem-limited bacteria:’Candidatus Liberibacter asiaticus’(CaLas),’Ca. L. africanus’(CaLaf) and ’Ca. L. americanus’(CaLam), of which CaLas is the most widespread and threatening one. All commercial citrus varieties or cultivars are susceptible to CaLas, which is transmitted both by grafting and insect vector, the citrus psyllid(Diaphorina citri, Trioza erytreae and Cacopsylla citrisuga). The most typical symptoms induced by CaLas are yellow shoots with leaf blotchy mottle and red-nose fruits, usually accompanied with leaf symptoms of zinc deficiency. Tristeza is also caused by a phloem-restricted pathogen, Citrus tristeza virus (CTV), a member of the Closterovirus of Closteroviridae, with the largest genome among known plant viruses. Like CaLas, CTV is transmitted by grafting and insect vector, the aphid (Toxoptera citricidus, T. aurantii, Aphis gossypii, A. spiraecola and Myzus persicae). CTV isolates can be divided into mild and severe types based on the severity of symptoms produced on citrus indicators. Mild CTV isolates cause no or mild symptoms on indicators, while severe CTV isolates cause three phenotypes, including quick decline (QD) of sweet orange on sour orange rootstock, stem pitting (SP) in sweet orange and grapefruit, and seedling yellow (SY) on sour orange. Simultaneous infection of trees with both CaLas and CTV is common in field. Symptoms caused by CaLas alone, or by severe isolates of CTV alone, or by CaLas plus severe isolates of CTV, are somehow similar, such as leaf yellowing, stiffening, and cupping, decline of roots and collapse of phloem tissue. Currently, the three most effective measures to reduce the spread of CaLas are to pull off the infected trees, to control the psyllids in a large scale, and to plant CaLas-free nursery seedlings. Curative chemicals and resistant varieties are unavailable for HLB so far, although hard efforts have been made for a century. Severe CTV isolates could be efficiently controlled through approaching the resistant varieties, or cross protection by pre-inoculation with mild CTV isolates.In this thesis, CaLas-infected Citrus sinensis tissues were observed by light and transmission electron microscopy (TEM). Biological symptoms of C. sinensis co-inoculated with CaLas and CTV were also observed. Pair-wise comparison of transcriptional profiles of C. sinensis infected with CaLas, and both mild and severe isolates of CTV, respectively, were carried out. These results could somehow facilitate our understanding of physiological changes and plant defense mechanisms employed in response to CaLas on the cellular level, and of the interactions between these pathogens and with C. sinensis on the whole plant level, and how these phloem-restricted pathogens affected plants, and how plants responded to these pathogens on the molecular level as well. The information obtained is also in favor of our insight into the molecular mechanisms of the pathogen-host interaction and biological symptoms through transcriptional molecular level. The knowledge will further improve the control of HLB through an understanding of natural resistance mechanisms to assist developing new ideas.Main results are as follows:1. Phage particles were observed in the CaLas-infected cells of C. sinensis for the first time in the world, and the observation of physical changes was made more systematically than ever before. The observation of CaLas-phage particles through TEM in this thesis is the first direct evidence for their existence in citrus. The phage type may be associated with the concentration of CaLas in C. sinensis plants. Starch grains and particles stained by osmium accumulated in and on chloroplasts, respectively. The former is related to carbohydrate metabolism, and the latter related to a biotic stress response. Callose that accumulated during plant defense, and idioblasts that contained calcium oxalate (CaOX) with dual functions to regulate Ca ion concentration and plant defense, were also observed. The accumulation of starch grains, callose and idioblasts, plant structures stained by osmium, indicated the imbalance of carbohydrate and Ca ion metabolism during the early stage when plant defense responses were activated in response to CaLas infection.2. The early establishment of mild CTV-B2in C. sinensis seedlings significantly reduced HLB incidence and symptoms. Comparisons were conducted between group I, in which trees were simultaneously inoculated with CTV-B2/CaLas-B232, severe CTV-B6/CaLas-B232and CTV-B2/CTV-B6, and group II, in which CaLas-B232was pre-inoculated, followed by CTV inoculation once confirmed the establishment of CaLas. We found that CTV established faster than CaLas in trees in group I, and the pre-establishment of CTV affected the following establishment of CaLas. This was especially the case with CTV-B2, which significantly reduced HLB incidence and symptoms, while pre-establishment of CaLas did not affect the following establishment of either mild CTV-B2nor severe CTV-B6. Simultaneous inoculation of CTV-B2/CTV-B6did not affect each other’s establishment, either. Early establishment of CaLas in rootlets rather than in young leaves was also confirmed, indicating that rootlets can serve as effective sampling part for early diagnosis of HLB.3. The number of differentially expressed genes (DEGs) in C. sinensis in response to the three pathogens reflected the complexity of symptoms caused by corresponding pathogens. Compared to mock-inoculated plants, significant changes were found in the transcriptome profiles in response to CTV-B2, CTV-B6and CaLas-B232, respectively. On the whole, more DEGs were up-regulated than down-regulated in response to all three pathogens. The number of differentially expressed transcripts in response to CaLas-B232(589up-regulated and22down-regulated) was about1.5times the number in response to CTV-B6(328up-regulated and76down-regulated), and about2times the number in response to CTV-B2(243up-regulated and42down-regulated). All three pathogens are phloem-limited, but the symptoms caused by CaLas-B232are the most complex, followed by CTV-B6, with leaf yellowing, stiffening and cupping and phloem collapse, while no symptom was observed by infection with CTV-B2. Therefore, the number of DEGs in response to pathogens was consistent with the complexity of symptoms caused by corresponding pathogens. The similarity of the transcriptome profiles of C. sinensis is consistent with the similarity of the biological symptoms in response to the three pathogens based on the number and GO categorization of DEGs. Among these DEGs,50transcripts were up-regulated, while only2transcripts were down-regulated in response to all three pathogens simultaneously. The number of differentially expressed transcripts in response to CTV-B6and CaLas-B232(105up-regulated and2down-regulated) simultaneously is approximately3.5times the number that responded to both CTV strains (27up-regulated and0down-regulated) or to combination I (CTV-B2and CaLas-B232)(22up-regulated and3down-regulated) simultaneously. GO categorization was conducted on DEGs by PlantGSEA, and the results showed that DEGs in response to CTV-B6and CaLas-B232were mainly involved in several biological process (BP) pathways, such as biotic stress responses, defense responses and signaling transductions, while DEGs in response to CTV-B2were primarily related to molecular function (MF) and cellular component (CC) pathways. As stated above, trees infected with CTV-B6or CaLas-B232alone show similar symptoms. Therefore, the transcriptome profiles revealed greater similarities of C. sinensis in response to CTV-B6and CaLas-B232than those to both two CTV isolates or combination I, which also reflected the similar symptomatic changes caused by CTV-B6and CaLas-B232.4. The circadian rhythm and ion balance of C.sinensis were heavily perturbed in response to all three pathogens, and many similar plant defense responses were triggered in response to all three pathogens but with different preferences. The up-regulation of PRR5, PRR7and GI genes indicated the circadian rhythm was greatly disturbed in response to all three pathogens, particularly to CTV-B6. The differential expression of Zn, Cu and Ca ions associated transporters manifested the imbalance of ion metabolisms in response to all three pathogens. Many similar plant defense responses were activated in response to the three pathogens but with certain preferences:1) Cell wall modification and secondary metabolism defense responses played more active roles in response to CTV-B2.2) Transcriptional related defenses responded more significantly to CTV-B6.3) Hormone-mediated, signaling transduction and R protein related defense responses functioned more notablely in response to CaLas-B232.5. The changes on the transcriptome level of C. sinensis in response to Ca Las is consistent with the observation by TEM. Genes related to callose synthesis and Zn ion transporters were differentially expressed, reflecting the callose deposition observed by TEM and Zn deficiency symptom on the leaves in response to CaLas.6. The results generated by Gfold were consistent with RT-qPCR results. Gfold is a program used to generalize fold change for ranking DEGs from RNA-seq data. It is designed based on the Poisson distribution, and the gene expression level is normalized as reads per kilo bases per million reads (RPKM), typically applicable for single biological data replicate.38DEGs were selected based on the fold change amplitude and gene function, and then assayed by RT-qPCR and calculated through relative expression method2-ΔΔCT. The Spearman’s coefficients between Gfold and RT-qPCR were0.82for CTV-B2,0.69for CTV-B6and0.81for CaLas-B232through Spss16.0calculation. The results indicated the good correlation between Gfold and RT-qPCR and the RNA-seq results obtained from Gfold are meaningful and reliable in the bioinformatic analyses.