| Huanglongbing (HLB), the most destructive. serious and uncontrollable citrus disease, is caused by a phloem-restricted bacteria, named Candidatus Liberibacter, Which can infect nearly all citrus species, cultivars and hybrids, as well as some citrus relatives. HLB has been an important harmful plant quarantine disease both at home and abroad. Neither HLB resistant rootstocks and citrus germplasm resources nor effective and durable HLB control methods have so far been found. The commonly used practices in controlling HLB spread are using disease free nursery materials and using chemicals to control its vector insect citrus psyllid in addition to eradicating the infected trees. Apparently, further researches on the plant-pathogen interaction mechanisms are needed to help us in understanding the host pathogenesis and in finding possible solutions to the disease such as potential resistance genes that could be used in breeding for HLB resistance.In this study. two citrus varieties(Citrus reticulata Blanco), Ponkan, a HLB sensitive variety, and Jiangxi red tangerine, one of the main rootstock in Guangdong province and seems to be HLB tolerable, were characterized for their reactions to HLB infection at transcriptomic or proteomic levels. Ponkan asymptomatic leaves from the early stage of HLB infection and symptomatic leaves showing blotchy mottle yellowing were used for the construction of a SSH (suppression subtractive hybridization) cDNA library, and were compared for their differences in gene expression patterns by Solexa sequencing of RNAs. Root samples of Jiangxi red tangerine were subjected to RNA-sequencing for profiling the differentially expressed genes (DEGs) and to isobaric tags for relative and absolute quantification (iTRAQ) analysis for detecting differentially expressed proteins. The expression patterns of some DEGs in these experiments were further confirmed by quantitative RT-PCR (qPCR). Results are shown as follows:1,A SSH library was successfully constructed using cDNA synthesized from RNA extracted from leaves of ponkan infected with HLB as tester and uninfected as driver. One hundred positive clones were randomly selected and sequenced. and71ESTs were obtained. A search based against NCBI GenBank, after removing the repeats and low quality sequences, revealed that41ESTs share considerable homology with known genes and that10ESTs did not have significant matches. Functional annotation of the genes showed that they were related to metabolic pathways and physiological and biochemical processes such as stress-tolerance, transportation, energy metabolism, photosynthesis, proteometabolism. signaling and anti-oxidation. It is noteworthy that the lectin protein precursor gene that is commonly induced by pathogen was found in the HLB bacteria-infected Ponkan leaf cDNA library. qPCR results showed that those two up-regulated genes were HLB induced. The above results indicated that an active anti-infection reaction was initiated in Ponkan leaves during the early stage of HLB bacteria infection.2,Ponkan leaves of13weeks and26weeks after HLB inoculation were used for digital gene expression analysis. BLAST search of the clementine unigene using the sequencing results were carried out. Some of the DEGs were confirmed with qPCR. Results indicated that:â‘ The number of up-regulated genes increased from37%in13wpi (weeks post inoculation) to64%in26wpi. what is more, the DEGs with fold change increased more than8times were increased from16.7%to87.3%. Gene ontology (GO) process molecular function enrichment analysis showed that the DEGs with oxidation reduction function increased from4.41%to8.48%and that DEGs responsive to stresses increased from1.10%to2.08%. but these related to defense responses decreased from0.74%to0.64%. However, those related to defense responses of down-regulated genes increased from0.55%to0.79%. Apparently, the expression level of resistance genes strengthened, while the defense ability of host declined along with enhanced stresses caused by HLB infection.â‘¡Photosynthesis related genes were down-regulated in both13wpi and26wpi, which indicated that HLB infection greatly reduced the citrus photosynthesis, perhaps via feedback regulation of the accumulated starches resulted from blockage of sieve tubes by the bacteria in the phloem tissue.â‘¢RIN4,a negative regulator in plant-pathogen interaction, was found up-regulated by approximately9-fold. The up-regulated expression of RIN4might play important roles in promoting bacteria growth.3,Jiangxi red tangerine root samples at50wpi were used in RNA-sequencing. Some DEGs were confirmed by qPCR. Results showed that:â‘ 3956genes were differentially expressed, among which65DEGs were identified with more than10-fold changes. DEGs were involving in19pieces of notable pathways, and the plant-pathogen interaction pathway related DEGs were enrichment very significantly.â‘¡RIN4was up-regulated about2.9times, which was much less than that in Ponkan, which may account for the difference in syndromes between the two cultivars.â‘¢SA signal transduction pathway related gene was up-regulated, the key gene of this pathway, NPR1was up-regulated1.6times, and the downstream PR1gene were up-regulated2.4times. JA signal transduction pathway related gene didn't have notable responses to HLB infection.â‘£Sucrose invertase was up-regulated1.7times, while the sucrose synthase were down-regulated1.8times. This may indicate that the sucrose level in HLB infected roots decreased as result of plugged phloem. Furthermore, ferredoxin expression was down-regulated2.2times, which may be related to nutrition deficiency.4,Comparative proteomic approach was also applied to decipher the pathogenic process of HLB in affected red tangerine roots using iTRAQ technique. Results showed that:â‘ The1445identified proteins mainly ranged from20to70kDa. Differentially expressed proteins were mainly involved in metabolism, defense reaction, antioxidation, transport and chitin metabolism.â‘¡36proteins showed high correlation with transcriptome results, and half of them were stress/disease resistance related, such as heat shock proteins, resistance proteins, peroxidase, miraculin-like proteins, chitinase, subtilisin-like protease and sieve element occlusion (SEO).â‘¢plugged phloem related protein SEO was up-regulated by1.67times, and this protein may function in inhibiting infection of the HLB bacteria and hampering the long distance transport of sucrose. Subtilisin-like protease was up-regulated with a fold change of282.09and the gene of this protein was also up-regulated3.14times, which suggested that the enzyme play important roles in inhibiting the infection of HLB bacteria and protecting the root meristematic zone. |
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