Differential Expression Proteomics Of Sweet Orang Induced By Severe And Mild Citrus Tristeza Virus Isolates

Citrus tristeza virus (CTV), a member of the genus Closterovirus within the family Closteroviridae, is widely distributed worldwide and is the causal agent of one of the most economically important diseases of citrus. CTV can cause great harm to sweet orange and pommulo trees. Multi-isolates have been found and cause a variety of symptoms such as decline and death of sweet orange and grapefruit on sour orange rootstock, stem pitting, seedling yellows, vein clearing etc.. CTV is dispersed to new areas by propagation of infection buds and then locally spread by aphids in a semipersistent manner. Simple application of Virus-free seedlings and chemical pesticides can’t entirely control the spread of CTV. Mild strain cross protection (MSCP) has been proven to be one effective way in preventing sensitive citrus varieties from damaged by some severe CTV strains. So far, little knowledge has been known on the CTV pathogenesis and mechanism of MSCP. Proteins are, in effect, the main actioners in cells and in an entire organism, which play animportant role in phosphorylation and glycosylation processes etc. More and more, the method of differential proteomics has been adopted to seek the pathogen-related proteins efficiently and sweet orange has been paid more attention due to its economic importance. Hence, the research on differential proteomics of CTV infection on sweet orange will make contributions to enhangce the understanding of its pathogenesis., and then further promote carryout of the CTV-resistant citrus breeding and developing the mild CTV strains, and in the end service the whole citrus industry.In this research, we established the suitable methods of total sweet orange(Citrus sinensis) leaf protein extraction and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).2D-PAGE and Two-dimensional fluorescence difference gel electrophoresis(2D-DIGE) combined with MALDI-TOF/TOF MS/MS techology were adopted to separate and analyse the differential expressed proteins induced by four CTV isolates, which included two severe isolates (TRL514and CT32), two mild isolates (CT30and CT11A). DeCyder software was used to accurately analyse the protein spots gained from2D-DIGE, which outputed the relative expression levels of protein spots, and Mascot software to search protein informations in NCBI database to blast the peptide sequences and gave out the matched protein accession IDs. KEGG databases were used to search out the corresponding KO number of each matched protein. We conducted three KEGG searches including Brite search, Pathway search and Module search to get the protein functional information. GO(gene ontology) analysis was adopted to find out gene products descriptions in terms of their associated biological processes, cellular components and molecular functions according to protein sequences. Then through integrated analysis based on above results, revealed the relationships between CTV isolates pathogenicity and induced protein expressions. In order to tentatively test the relations between CTV isolates and induced protein expressions, Realtime RT-PCR techology was adopted to detect differential genes expression in CTV-infected and healthy sweet orange leaves. Followed are the main results.1. Establishment of2D method of sweet orange leaf proteinThree protein extraction methods were compared, including phenol method, trichloroacetic acid and acetone precipitation method, trichloroacetic acid and combined phenol method, and SDS-PAGE was run to test. Result showed that the trichloroacetic acid and combined phenol method was the most suitable protein extration method for sweet orange leaves. Isoelectrofocusing experiments showed that focusing parameters from gel instructions couldn’t be adopted directly and should find out the suitable program for your samples. In our study, suggested conditions of isoelectrofocusing were:8000V and30-100kVhr for7cm gel;8000V and80-150kVhr for13cm. Combined2D-MALDI-TOF MS analysis vestified the validity of this2D method.2.2D electrophoresis of differential expressed proteins in sweet oranges induced by four CTV isolatesTwo sweet orange varieties including Jincheng and Symons, four CTV isolates including two severe isolates (TRL514and CT32) and two mild isolates(CT30and CT11A) were adopted in this experiment. Through2D method, we found that most differential expressed protein spots located within ph4-7area. To Jincheng seedling samples, seperated20differential expressed spots in pH3-10,18cm gels, wheras42spots in pH4-7,13cm gels. It indicated that more protein spots could be seperated in pH4-7,13cm gels. After that, we seperated35spots from Jincheng budling samples, and53spots from Symons budling samples.3.2D-DIGE analysis of differential expressed proteins in sweet oranges induced by severe and mild CTV isolates.2D-DIGE is the representive gel quantitative techology for differential expressed protein analysis. In this study, we used DIGE method to analysis the differential protein expression induced by one severe CTV isolate(TRL514) and one mild CTV isolate(CT11A) compared to healthy Control. The results showed that total82differential protein spots were detected with one-way analysis(0≤p≤0.001) between three groups. And when employing relative average volume ratio≥1.2or≥1.5statistical variance of two groups(t-test,P<0.05)respectively,84or32differential protein spots were detected between severe and mild isolate, and139or91spots between severe isolate and control, and116or84spots between mild isolate and control. Altogether up-expressed protein spot nubmers were more than down-expressed, indicating the reality of more protein expressions happening in CTV-infected sweet orange plants.4. CTV-induced differential expressed protein analysis using MALDI-TOF/TOF MS/MS and protein function searchesThe CTV-induced differential expressed proteins were identified by4800Plus MALDI TOF/TOFTM Analyzer mass spectrometer produced by ABI company. Results showed a total of76non-repeated proteins were identified successfully from140DIGE protein spots. Approximately two times of acid protein(all pI ranged between pH4-7) spot number to basic protein(most pI ranged between pH8-9). And most proteins ranged between10-55kDa.KEGG analysisApplication of KEGG blast search indicated that54identified proteins belonged to27KO types. Further Brite search results showed that all27KO proteins could be classified into8classes. In which, over20KO proteins classified into protein ontology and enzymes, that indicated CTV-induced expressed proteins took part in many biological proccesses. Pathway clustering results showed quite a number of proteins affected two specific pathways, that were neurodegenerative diseases pathway and folding、sorting and degradation pathway. The related KO proteins contained K13993、K02730、K02732、K01689、K00134and K04565, which might play some key roles in affecting the host protein expression when diseased with CTV.To learn how the27KO proteins behave in pathway, pathway and module searches were conducted. Results indicated all27protein types involving30pathways and21functional module, or having quite more functions.GO analysisThrough Gene Ontology analysis of these76identified proteins, we found that CTV-induced expressed proteins mainly located in cell, cell part, organelle and membrane, which had catalytic activity and binding activity. There were a number of proteins located in extracellular region and macromolecular complex which had antioxidant activity, enzyme regulator activity and transporter activity, attending the processes of biological regulation, response to stimulus and localization. It indicated the referred proteins’close relation to CTV pathogenesis.Combined analysis of KEGG and GO ontology suggested10differential expressed proteins may play the key roles in response to CTV infection, including glyceraldehyde3-phosphate dehydrogenase, Cu/Zn superoxide dismutase, HSP20family protein, enolase,20S proteasome subunit alpha1,20S proteasome subunit beta6, isocitrate dehydrogenase, F-type H+-transporting ATPase subunit beta, Thioredoxin H-type5.5. Comparison of differential protein expressions in sweet orange induced by severe and mild CTV isolatesBased on above analyzations, we compared the protein expression differences in sweet orange induced by severe and mild CTV isolates. Results showed that there were97differential protein spots betweet TRL514isolate(severe) and Control(healthy) found in123identified protein spots, among which61differential protein spots were up-regulated compared to Control. And89spots between CT11A(mild) and Control, among which61up-regulated compared to Control. And51spots between TRL514and CT11A,among which24up-regulated compared to CT11A.There were14differential protein spots corresponding to the10key protein types. And6kinds of proteins including glyceraldehyde3-phosphate dehydrogenase(K00134),20S proteasome subunit alpha1(K02730),20S proteasome subunit beta6(K02732), F-type H+-transporting ATPase subunit beta(kO2112), Cu/Zn superoxide dismutase(K04565) and thioredoxin H-type5(gi|357438577) were up-regulated in severe or mild CTV-infected sweet orange compared with healthy control, which usually play an important role in the immune system. And3kinds of proteins including HSP20family protein(K13993), fructose-bisphosphate aldolase class I(k01623) and isocitrate dehydrogenase (k00031) were down-regulated in severe or mild CTV-infected sweet orange compared with healthy control, which play a role in signal transduction.6. Examination of mRNA expression induced by CTV using RT-qPCR techologyFor the further examinaton of regarding protein expression relations, four differential protein genes expressions in TRL514-inoculated, CT11A-inoculated and healthy control Jincheng leaves were detected by RT-qPCR respectively. Tow citrus house-keeping genes were used imutaneously to balance the primary sample templates and adoption of relative expression level to compare the difference between samples. Results showed only one gene expression be verified the same orientation with that from DIGE. But results also supported great differences in protein expressions between severe and mild CTV isolates inductions.