Proteomics analysis of early pathogens after infection of Candidatus Liberibacter asiaticus with Jincheng Orange (Citrus sinensis Osbeck)

Abstract: 【Objective】Huanglongbing (HLB) is the most devastating disease in citrus production throughout the world. Because Candidatus Liberibacter asiaticus (CLas), the most virulent pathogen of HLB identified at present, cannot be cultured in vitro, its main pathogenic mechanism is still unrevealed. Therefore, proteomics analysis was used to explore the expression of pathogenic proteins during the early CLas infection stage in Citrus sinensis Osbeck.【Methods】In this study, the leafdisc grafting was used to infect the healthy Jincheng Orange (Citrus sinensis Osbeck) to transmit the CLas. Twomonths later, the CLas was detected by PCR, then the infected roots and leaf veins were collected for iTRAQ proteome Sequencing. The sequencing result was annotated while the genome of the CLas was used as a reference, and the infected leaf veins were used as control to screen the proteins of the CLas which expressed differently. Finally, qRT-PCR was used to verify those significantly expressed differently proteins.【Results】A total of 38 pathogenic proteins were identified, 13 pathogenic proteins were  up- regulated and 14 pathogenic proteins were down- regulated while there were 7 pathogenic proteins with unknown functions. The proteome of the infected roots was screened, the infected leaf veins were used as control, the difference fold value more than 1.5 times were used as a significant up-regulation and less than 1/1.5 as a significant down- regulation standard. As a result, there were 2 proteins (ACT56611. 1. ACT66821.4) were significantly up- regulated, and 4 proteins were significantly downregulated (ACT56726.1, ACT56912.1, ACT57006.1, ACT57090.1). Gene ontology (GO) analysis was performed on the 38 identified pathogenic proteins. The results showed that pathogenic proteins were mainly involved in 10 biological processes, but were mainly related to nucleic acid binding and material transport activities. All pathogenic proteins were involved in 10 molecular functions and there were 22 related to the cellular biosynthetic process, accounting for 57.9% of the total proteins, 12 pathogenic proteins related to the cellular macromolecule biosynthetic process, accounting for 31.6% of the total proteins. Among 38 pathogenic proteins, 11 pathogenic proteins were related to the molecular function of ribonucleotide binding, accounting for 28.9% of the total proteins, 5 pathogenic proteins were related to transporter activity, accounting for 13.2% of the total proteins. The 38 pathogenic proteins were used to construct a protein interaction network diagram which showed that more proteins interacted with ACT57362.1, ACT56735.1, ACT57090.1, ACT57110.2, and ACT56789.1. The protein annotation clarified that ACT57362.1 was inosine 5’-monophosphate dehydrogenase, which catalyzes the conversion of inosine 5’-phosphate (IMP) to xanthine 5’-phosphate (XMP). It is the first important and rate-limiting step in the de novo synthesis of guanine nucleotides, and it plays an important role in regulating cell growth; ACT56735.1 was the 30S ribosomal protein S10, which is involved in the binding of tRNA and ribosomes; ACT57090.1 was the alpha subunit of ATP synthase, which converts ADP to ATP in the presence of a proton gradient on the membrane; ACT57110.2 was the chaperone protein DnaK, which plays an important role in the process of protein folding. ACT56789.1 was the β subunit of phenylalaninetRNA ligase. The results of these 5 pathogenic proteins annotations showed that the key proteins related to growth and development of cell and protein folding were expressed to response to the CLas at the early HLB infection stage in Jincheng Orange. The result of qRT-PCR showed that the expression of 6 significantly differentiated proteins(ACT56611.1, ACT66821.4, ACT56726.1, ACT56912.1, ACT57006.1,ACT57090.1)in the roots were significantly higher than that in the leaves; the expression of CLasMalE (ACT56611.1) transporter proteins, an important component of the ABC transport system, is as high as 595 times. The bioinformatics analysis of ClasMalE showed that its open reading frame was 1122 bp and there was 1 exon but no intron, so it encodes 373 amino acids. The protein primary structure prediction showed that the molecular weight (MW) of the protein was 4.4 ku, and the theoretical pI was 9.02,so it was a hydrophilic protein. The secondary structure of the CLasMalE mainly included α-helix, random coil, extended strand and beta turn. The conserved domain prediction revealed that its protein sequence contained a MalE domainand its number was COG0767. Finally, this study analyzed the expression characteristics of the CLasMalE in citrus, it showed that its expression in the roots of susceptible species was significantly higher than that in the leaf veins, and the expression in the old leaves was significantly higher than that in the young leaves, but there was no significant difference in tolerant species sour pomel【Conclusion】The high-level expression of ABC transport system’s important component ClasMalE in the infected Jincheng roots was found, indicating that the ABC transport system would play an important role in the host infection of the CLas.