Proteomic analyses of Citrus petiole responses to early Huanglongbing Disease

Backgroud Huanglongbing (HLB) is currently one of the most destructive citrus disease worldwide. It is caused by Candidatus Liberibacter asiaticus (CLas), a nonculturable alpha-proteobacterium, which it resides exclusively in the phloem tissues. Therefore, understanding the early CLas-responsive proteins in citrus petiole where pathogenic bacteria colonized will help to investigate plant resistance to the pathogen.Results In this study, a comparative proteomic approach was applied to identify the petiole proteins associated with the response to CLas infection. A total of 777 proteins were differentially expressed in response to CLas. Among them, 499 proteins were up-regulated and 278 were down-regulated. Among the most highly up-regulated differentially expressed proteins (DEPs) were salicylate carboxymethyltransferase, ubiquitin carboxyl-terminal hydrolase 13, trans-resveratrol di-O-methyltransferase, linoleate 13S-lipoxygenase 2-1, granule-bound starch synthase 1 and thaumatin-like proteins. While the most highly down-regulated DEPs were oxygen-evolving enhancer proteins, ribulose bisphosphate carboxylase/oxygenase activase, peroxidases and photosystem reaction center subunits. The results of qPCR analysis of a number of indicated DEPs and western blotting further validated four representative DEPs, including salicylate carboxymethyltransferase, linoleate 13S-lipoxygenase 2-1, granule-bound starch synthase 1 and photosystem I reaction center subunit showed that most of detected DEPs were positively correlated with their mRNA and protein levels.Conclusions Our comparative proteomic analysis rst proling reveals early and primary proteome alterations in CLas-infected citrus petiole, where pathogens reside in. The DEPs results demonstrate that CLas infection could promote the carbohydrate metabolism, depress the photosystem and activate/inhibit defense responses.

proteins. While the most highly down-regulated DEPs were oxygen-evolving enhancer proteins, ribulose bisphosphate carboxylase/oxygenase activase, peroxidases and photosystem reaction center subunits. The results of qPCR analysis of a number of indicated DEPs and western blotting further validated four representative DEPs, including salicylate carboxymethyltransferase, linoleate 13S-lipoxygenase 2-1, granule-bound starch synthase 1 and photosystem I reaction center subunit showed that most of detected DEPs were positively correlated with their mRNA and protein levels.Conclusions Our comparative proteomic analysis rst pro ling reveals early and primary proteome alterations in CLas-infected citrus petiole, where pathogens reside in. The DEPs results demonstrate that CLas infection could promote the carbohydrate metabolism, depress the photosystem and activate/inhibit defense responses.

Background
Huanglongbing (HLB), which is also called citrus greening disease, is a destructive citrus disease worldwide [1]. It is caused by a gram-negative and phloem-inhabiting bacterium of the genus Candidatus Liberibacter, including three known species, "Candidatus Liberibacter asiaticus (CLas)", "Candidatus Liberibacter americanus" and "Candidatus Liberibacter africanus". All known citrus species and citrus relatives can be infected with the pathogens. HLB disease is rst reported in Asia and is widely distributed in more than 40 countries and regions in Asia, Oceania, Africa, and the Americas [2][3][4][5]. The HLB pathogens could be transmitted by the citrus psyllid Diaphorina citri [6,7]. After colonizing citrus trees, the pathogen spreads to all tissues quickly, and leads to plant death after a period of several months to a few years [8]. The pathogen blocks the phloem of citrus trees, resulting in symptoms such as vein yellowing and hardening, and blotchy mottling and chlorosis that are sometimes mistaken for element de ciency.
The fruits of infected trees are small, with a bitter and acidic avor as well as a smooth but lackluster pericarp [8][9][10].
It is important to clarify the host responses to CLas infection. Previous studies have been reported to identify CLas-affected genes and proteins in leaf, root and fruit [11,12]. These studies suggested that CLas could mediate a lot of signi cant biological processes in plants, including carbohydrate metabolism, photosynthesis, multiple hormone pathways and secondary metabolites. Previous studies also have showed that there were remarkable differences between diseased and healthy fruit in the concentrations of sugars, limonin glucoside and some other organic compounds [13]. These differences explained the bitter and acidic avor of the fruit from diseased trees. CLas-infection depressed the absorption of elements in roots and leaves [12], this is one reason why CLas-infected leaves show several symptoms of element de ciency. Remarkably, the carbohydrate transport system is largely disturbed by CLas. Studies suggested that phloem blockage in infected trees caused starch accumulation in plastids and phloem [14][15][16]. Excessively accumulated starches results in disruption of chloroplast inner grana structure [17].
Transcriptomic works have been demonstrated that genes related to starch metabolism and photosynthesis were down-regulated in all detected tissues [11,18,19], however, photosynthesis-related genes were up-regulated in fruit [20]. Additionally, CLas was able to enhance transcription of genes involved in photosynthesis and ATP synthesis [21]. These transcriptomics data are to some extent consistent with several reported proteome results. Photosynthesis-related proteins, such as oxygenevolving enhancer (OEE) proteins and a photosystem I 9 kDa protein, were down-regulated in CLasinfected plant leaf tissues, however, several proteins related to starches metabolism were found to be upregulated [22]. Although many proteins related to carbohydrate metabolism and photosynthesis were identi ed in a up/down-regulation manner, in addition to validation in transcriptional levels, no further con rmation was performed.
The PP2-like genes were observed to be signi cantly up-regulated in CLas-infected roots [12], implying a possible defense response induced by CLas against psyllids, although no observation of regulated PP2 in leaf and other tissues. The most interesting point is the plant innate immune system mediated by hormonal crosstalk, particularly between salicylic acid (SA) and jasmonic acid (JA) pathways. SA and JA signaling pathways were activated in CLas-infected plants as reported previously [12,26], and CLas also mediates ethylene, auxin and brassinosteroid signaling pathways [27].
Previous studies generally focused on the differentially expressed genes or proteins (DEPs) mediated by CLas in leaves, fruits and roots [26,[28][29][30][31][32]. Leaves infected with CLas display yellowing and blotchy mottled appearances. However, the pathogens colonize in the phloem. The global analysis of infected source tissues is an appropriate tool to understand the fundamental effects of CLas in plants. Here, a systematic analysis of the citrus petiole proteome was performed through the use of a comprehensive proteomic method to explore global changes in protein expression in response to CLas infection.
Furthermore, qPCR and western blotting were performed to validate the reliability of proteome data. To best of our knowledge, this is the rst study focusing on the proteome of CLas-infected citrus petiole, where pathogens reside in. The results provide a new insight into knowing fundamental and initial responses of citrus to CLas infection.

Results
Characterization of the proteome of the CLas-affected and control citrus petiole HLB symptoms appeared at 4 months post CLas-inoculation, containing yellowing, blotchy mottle and/or variegated chlorosis of leaves (Fig. 1A). The presence of CLas was con rmed by qPCR. Ct values of CLas-infected petioles were less than 30 while control samples showed no ampli ed product (Fig. 1B).. To examine the variation in the proteome of citrus petioles infected with CLas, a proteomic method based on LC−MS/MS was applied and tandem mass spectra were searched against the UniProt_Citrus sinensis database. Total protein was extracted from petioles of mock-inoculated (without symptoms, qPCRnegative) and CLas-inoculated (with symptoms, qPCR-positive) sweet oranges as described [33]. In our study, a large number of peptides were identified based on the MS data. The length of most peptides distributed was between 6 and 14 amino acids, which agrees with the property of tryptic peptides ( Fig.  2A).. The distribution of peptide delta mass in each replicate is between -1.2-2.2 ppm (Additional le 1: Figure S1),, and the number of spectrum for each protein in different replicates showed a reasonable consistency (Additional le 2: Figure S2),, which means the mass accuracy of the MS data t the requirement. The analysis of LC-MS/MS data showed the identi cation of 7321 proteins (False Discovery Rate (FDR) was set as 1.0%, at least 2 unique peptide for one protein) (Additional le 3: Table S1),, of which 5270 proteins were identi ed in each biological replicate (Fig. 2B).. Further analysis identi ed 777 proteins as DEPs at a cutoff value of >|±1.3|-fold (p value <0.005), which included 499 up-regulated and 278 down-regulated proteins ( Fig. 2C and Additional le 3: Table S1).. Of the DEPs, a salicylate carboxymethyltransferase (CsSAMT, A0A067FUD3) was signi cantly up-regulated by more than 3-fold. The ubiquitin carboxyl-terminal hydrolase (A0A067DGD4) was increased by 2.95-fold. A trans-resveratrol di-O-methyltransferase (A0A067DIU3) and linoleate 13S-lipoxygenase (CsLOX2.1, A0A067ERJ6) showed a 2.43 and 2.23-fold increases, respectively. In addition, a histone deacetylase HDT1 and two oxygenevolving enhancer proteins were found to be down-regulated by more than 2-fold.

Gene ontology (GO) analysis results
To further study the proteomics data, a GO analysis was performed to classify the DEPs to account for their biological processes, molecular functions and cellular component ( Fig. 3A and Additional le 4: Table S2).. In the biological process category, there were 145 GO terms, the protein groups associated with response to stimulus (GO:0050896), single-organism metabolic process (GO:0044710) and response to stress (GO:0006950) accounted for 17.25%, 14.67% and 11.58%, respectively, indicating that response to stimulus and single-organism metabolic process are likely to be affected signi cant by HLB-infection. The organism's response to stimulus suggested that some proteins participated in the perception and transmission of extrinsic stimuli signals or that the proteins may be conducive for plants to perceive external stimuli. At molecular function GO level, 60 total GO terms were assigned, the protein groups connected with catalytic (GO:0003824), hydrolase activity (GO:0016787) and cation binding (GO:0043169) accounted for 27.67%, 10.42% and 9.91%, respectively. The other important protein groups on molecular function were oxidoreductase activity (GO:0016491) and transporter activity (GO:0005215). In the cellular component category, there were 81 GO terms, corresponding to 35.39% of proteins in the cells (GO: 0005623), 32.56% of proteins in the intracellular (GO:0005622), 29.73% of proteins in the cytoplasm (GO:0005737) and 28.44% of proteins in the organelle (GO:0043226). The metabolic processes in biological processes and the catalytic activity in molecular functions showed that DEPs are associated with enzymatic proteins involved in metabolism.

KEGG pathway analysis
To determine the involvement of these DEPs in response to CLas-infection, a pathway analysis was carried out to identify the potential target proteins. The DEPs have been identi ed to be involved in 5 main pathways ( Fig. 3B and Additional le 4: Table S2),, including photosynthesis (cit00195), protein processing in endoplasmic reticulum (cit04141), phenylpropanoid biosynthesis (cit00940), galactose metabolism (cit00052) and monobactam biosynthesis (cit00261). Among them, the largest number of DEPs were enriched in protein processing in endoplasmic reticulum, followed by the phenylpropanoid biosynthesis and photosynthesis, respectively. These data clearly indicate that HLB has signi cant regulatory effects in citrus petiole cells.

Analysis and veri cation of carbohydrate synthesize related DEPs
In CLas-inoculated plants, there was a lot of CLas-mediated down-regulation DEPs. Remarkably, 21 DEPs were involved in carbohydrate metabolism, most of which were up-regulated in CLas-inoculated plants compared to mock-inoculated plants (Additional le 5: Table S3),, especially in D-glucose and starch synthesis pathways (Fig. 4A).. Additionally, to explore the regulation in transcript levels, 10 representative DEPs were chosen to perform qPCR to validate the gene expression patterns. The transcript level in the Mock-inoculated plant was set to 1.0 (black square) and the CLas-inoculated fold-regulation ratios were shown in Table 1. The results indicated that the expression pro le of all detected genes was in agreement with the proteome data. In addition, an indicated fragment of the protein (Granule-bound starch synthase 1, CsGBSS1, A0A067H3M6) was rst expressed in E. coli (Fig. 4B),, the puri ed protein was then used to prepare its antibodies in rabbit. Western blotting was then carried out to con rm the translational levels of CsGBSS1 with its antibodies. The result exhibited a similar mediation with proteome result in response to CLas (Fig. 4C).. These data indicate that CLas-infection indeed induced an up-regulated expression of starch and sucrose metabolism-related proteins. Table 1. Representatives of differentially expressed proteins on starch and sucrose metabolism identi ed in CLasinfected leaf petioles. falseAnalysis and veri cation of photosystem-related DEPs According to analysis of KEGG enrichment, there were total 18 DEPs involved in citrus photosynthesis in response to CLas-infection (Additional le 5: Table S3).. CLas-inoculated plants used in this study showed typical blotchy mottling and chlorosis symptoms. We observed that these DEPs were involved in photosystem I (4 proteins, A0A067FZ61, A8C1A6, A8C172 (CsPSI) and A0A067ESN4), photosystem II (7 proteins, A0A067F2K2, A0A067GIE1, A0A067DPL0, A0A067FX28, A0A067DU68, A0A067GJW2 and A0A067EDZ9), photosynthethic electron transport (3 proteins, A0A067HB98, A0A067DS64 and A0A067E882) and F-type ATPase (A0A067H8B2) (Fig. 5A), which are the components of the photosystem (Additional le 6: Figure S3, http://www.genome.jp/kegg-bin/show_pathway?cit00195). Additionally, a Rubisco (A0A067EBB3), carbonic anhydrase (A0A067FFU3) and protein disul de-isomerase (A0A067HAZ0) were observed in a decrease manner in CLas-infection plants compared to mock plants.
To validate the proteome data, 10 proteins were con rmed using the qPCR in transcript levels. As shown in Table 2, the expression pro les of 7 tested genes were consistent with the proteome results, showing a down-regulation in mRNA levels. While the transcriptional levels of carbonic anhydrase 2 (A0A067FFU3), protein disul de-isomerase (A0A067HAZ0) and ATP synthase delta chain (A0A067H8B2) indicated a not signi cant regulation. At the meantime, the antibodies of CsPSI was prepared to perform western blotting analysis, the result showed that CLas decreased the level of CsPSI in plant ( Fig. 5B,C).. In addition, we found that the expression of chlorophyllase(A0A067DGV8) was signi cantly increased by 1.67 times(Additional le 5: Table S3). Table 2. Representatives of differentially expressed proteins on photosystem identi ed in CLas-infected leaf petioles.

falseAnalysis and veri cation of defense responses related DEPs
CLas-infection resulted in a series of variation of the plant defense-related proteins (Additional le 5: Table S3).. Several hormone-related proteins were differentially expressed in CLas-infected plants, including a CsSAMT and a CsLOX2.1, which were signi cantly up-regulated 3.46 and 2.23-fold, respectively. In addition, SA-mediated defense-related proteins, CsPR1 (A0A067DC18) and CsPR5s (A0A067FP42 and A0A067DCM7) (also called thaumatin-like protein), were all up-regulated (Table 3 and Additional le 5: Table S3).. Interestingly, most of the DEPs involved in phenylpropanoid biosynthesis were down-regulated by CLas-inoculation (Fig. 6A).. The expression of 14 genes in citrus petioles was validated by qPCR analysis to corroborate the proteome data. The results were shown in Table 3, revealing a consistency with proteome and transcript data. As the expression of SA and JA-related proteins (CsSAMT and CsLOX2.1, respectively) was elevated in a high level in response to CLas infection, we further con rm their expression level via western blot analysis (Fig. 6B,C),, the results suggested that CLas caused the accumulation of CsSAMT and CsLOX2.1. Furthermore, the content of SA and JA in both CLas-inoculated and mock-inoculated plants were detected, the data showed that the SA and JA contents were indeed elevated by CLas-infection (Fig. 6D).. Table 3. Representatives of differentially expressed proteins on plant defense response identi ed in CLas-infected leaf petioles.

Discussion
HLB is a devastating citrus disease that causes tremendous economic losses to the citrus industries worldwide. It is of di culty to control, one of the reasons is that the pathogen is a phloem-restricted bacterium. the rst global analysis of citrus petiole protein pro les using TMT-labeled technology was carried out in this study. A total of 777 proteins were differentially expressed in response to CLas infection. The CLas-mediated DEPs involved in synthesis of carbohydrate, photosystem and defense responses of plants mediated by CLas-infection were mainly discussed based on our ndings.

CLas-infection enhances the synthesis of carbohydrate
The accumulation of starch in CLas-affeted plant tissues has been previously reported [15,16]. A study has been shown that the accumulation of starch is assumed to be resulted from phloem plugging associated with not only the bacteria growing inside but also callose deposition and accumulation of phloem proteins [17]. We found that the up-regulation of starch synthesis-related proteins may be direct reason for its accumulation. These results correspond to the previous studies that showed an upregulation of starch metabolism-related genes/proteins [12,22], although starch metabolism genes were also down-regulated in several examined tissues [21,34].
In general, the surplus carbohydrates produced during photosynthesis would be stored in form of starch in plants. Starch is a polymeric carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. It consists of two types of molecules: the linear and helical amylose and the branched amylopectin. Plants produce starch by rst converting glucose 1-phosphate to ADP-glucose using the enzyme glucose-1-phosphate adenylyltransferase. The starch synthase then adds the ADP-glucose via a 1,4-alpha glycosidic bond to a growing chain of glucose residues, liberating ADP and creating amylose. Starch branching enzyme introduces 1,6-alpha glycosidic bonds between these chains, creating the branched amylopectin [35]. A ADPase (glucose-1-phosphate adenylyltransferase, A0A067DG91), 3 starch synthases (A0A067H3M6 (CsGBSS1), A0A067ESR0 and A0A067GDC7), 2 glucan-branching enzymes (A0A067F4L8 and A0A067HB75) were up-regulated in CLas-inoculated plants, and the CsGBSS1 was upregulated to 2.23-fold (Table 1 and Additional le 5: Table S3).. However, ADPase, which is rate-limiting enzyme for starch biosynthesis, has been shown to be repressed in CLas-infected plants [26]. Glucose-6phosphate/phosphate translocator 2 (GPT2) is involved in the formation of starch by transporting glucose into chloroplast [36], and has been reported to be decreased by CLas [21]. We identi ed a signi cant up-regulation (1.72-fold) of GPT2 (A0A067EH66) in protein level. These results suggest that starch biosynthesis-associated enzymes may be up-regulated during the early disease development of HLB, which agrees with several previous works [14,22,37]. Here, CsGBSS1 with high induced change fold was chosen to prepare its antibodies, and the western analysis con rmed that this protein was indeed upregulated in response to CLas infection (Fig. 4C).. This is the rst time to validate the result via western blotting analysis, particularly in citrus petiole.
The photosystem was signi cantly suppressed by CLasinfection CLas disturbs the metabolic system of plants and results in yellowing and blotchy on leaves [38], according with the striking results that all photosynthesis-related proteins identi ed here showed downregulation, except that the expression of chlorophyllase(A0A067DGV8) was signi cantly increased by 1.67 times(Additional le 5: Table S3). This manifests that pathogens can also indirectly degrade chlorophyll by inducing plants to produce chlorophyll enzymes. It was the rst time that we used proteomics to found the chlorophyllase was induced upregulation in citrus petiole where were directly parasitized by CLas, and eliminated interference from other tissues that are not directly in contact with the pathogen. Combined with Western blot and proteomics data, it is assumed that photosynthesis was inhibited not olnly by destroying the PSI reaction center subunit II(A8C172), but also directly promoting the synthesis of chlorophyll hydrolase(A0A067DGV8) in the early stage of HLB infection. These results suggest that CLas-infection had signi cant negative effects on citrus photosystem by regulating the CsPSI and chlorophyllase in early infection stage.
Rubisco is an enzyme involved in the rst major step of carbon xation and catalyzes the carboxylation of ribulose-1,5-bisphosphate [39], and it in general is used as a housekeeping protein in study. Agree with previous studies, Rubisco has been shown to be depressed in response to CLas-infection [11,22]. The possible explanations of the reduction of Rubisco are that the accumulation of starch and sucrose metabolism suppresses its production, or the de ciencies of nutrients causes its degradation [40]. 3 oxygen-evolving enhancer (OEE) proteins and a PSII 10 kDa polypeptide, which are involved in PSII oxygen evolving complex assembly, were observed a more than 1.5-fold decrease ( Table 2 and Additional  le 5: Table S3).. OEEs are involved in splitting water into proton and oxygen molecules, and are required for PSII assembly/stability [41,42]. OEEs have been shown to stabilize the manganese cluster [43], although a previous study showed that the manganese content was not signi cantly reduced in CLasinfected plants [22]. Citrus plants with Mn-de ciency also display symptoms, including reticular light/dark green and chlorosis of leaves, which are similar with CLas-infected plants. Reduced Mn is available form for plants, it has been demonstrated that pathogens can oxidize Mn to oxidized form, which is unavailable for plants [40]. Decreased OEEs may not stabilize the Mn in available form, resulting in Mnde ciency symptoms without decrease of its content. The DEPs in PSI were mainly involved in forming complexes with ferredoxin (Fd) and ferredoxin-oxidoreductase (FNR), and assisting the docking of the Fd to PSI and interaction with FNR. Fd/FNR oxidation-reduction system has effect on the production of NADPH, which further affects the processes of carbon xation [44]. We showed here that the level of several PSI-related proteins including CsPSI was reduced in CLas-infected plants ( Table 2 and Fig. 5C).. This seems to contradict the up-regulation of carbohydrate metabolism discussed above, thus how CLas mediates photosystem and carbohydrate metabolism needs further study.
The up-regulation of starch synthesis-related proteins in plant tissues during CLas infection has been previously reported [12,22,26,37] and we earlier showed that CLas-induced up-regulation of starch and sucrose metabolism-related proteins occurred in the petiole where pathogens reside in. The accumulation of photosynthates could inhibit the photosynthesis by suppressing photosynthetic activity via a negative feedback mechanism [45]. Our ndings indicated that CLas could proactively promote starch synthesis to depress photosystem indirectly and directly in the early infection stage, suggesting that the activity of the PS might be one of early targets during HLB disease development. Increasing studies have demonstrated that chloroplast and photosynthesis play important roles in plant defense responses, including production of reactive oxygen species (ROS), reactive nitrogen oxide intermediates (NOI), hormones SA and JA [46][47][48][49][50].

Effects of CLas-infection on plant defense responses
Signi cantly, a lot of hormone-related proteins were differentially expressed in plants in response to CLas.
SA plays important roles in plant defense and in activating systemic acquired resistance (SAR) [51][52][53]. Bacterial PAMPs, such as g22, can induce SA accumulation. Previous study has been reported that CLas can also encode a 452-amino acid agellin protein containing a conserved 22 amino acid domain ( g22) [54]. This would be one reason for the increase of SA production in CLas-infection plants (Fig. 6D).. In addition, a protein (A0A067FR53) involved in the EDS1-dependent intrinsic and indispensable resistance signaling pathway was elevated 1.59-fold (Additional le 5: Table S3).. EDS1 is able to encode a lipase that modulates SA-dependent disease resistance [55]. The SA-mediated CsPR1 and CsPR5 proteins showed a similar regulatory pattern with SA-related proteins (Table 3).. And these PR proteins have been reported to be induced in response to pathogen attack and are associated with disease resistance and SAR [56,57]. Interestingly, JA signaling pathway was also activated by CLas-infection. In addition to CsLOX2.1, many DEPs related to the JA pathway, including 4 glutathione S-transferases and a 4coumarate-CoA ligase (Additional le 3: Table S1),, were induced while some of them were reduced. The SA/JA content in CLas-infected petioles was indeed relatively higher than that in mock petioles (Fig.  6C,D).. These results indicate that the SA and JA pathways were induced in this study, which is different from a previous study [37]. Generally, JA pathway is required for resistance of plants against necrotrophic pathogens, while SA signaling pathway is activated against biotrophic pathogens, and JA can antagonize SA signaling [58]. CLas is a biotrophic pathogen, the activation of JA may antagonize the content of SA level to some extent, contributing to HLB disease development. A recent study has reported that CLas is able to encode a SA hydroxylase to degrade SA, contributing to disease development [59]. http://www.freepatentsonline.com/y2017/0073700.html). The present invention relates to transgenic citrus trees resistant to HLB through overexpression of AtNPR1, and the results demonstrated that overexpressing the AtNPR1 can result in effective HLB resistance in citrus. NPR1 is a key regulator in the signal transduction pathway that leads to SAR, and transduction of the SA signal requires the function of NPR1 [60]. It would be an important research to clarify how CLas mediate SA and JA crosstalk during its infection. In fact, CLas affected multiple hormone-mediated immune responses. A auxin (A0A067ETE6)induced protein was up-regulated, suggesting that the auxin-regulated pathways were activated by CLas infection. Moreover, several DEPs involved in the signal transduction of abscisic acid (A0A067FB99) and brassinosteroid (A0A067FLJ4) were also induced.
Furthermore, Secondary metabolites play signi cant roles in disease resistance, including phenylpropanoid-containing substances based on different biosynthesis pathways [61]. Phenylpropanoids are found to serve as essential components of a number of structural polymers and to defend against pathogens [62]. It has been demonstrated that ligni cation and reinforcement of cell walls are important processes of plants in response to pathogen infection [61,63]. Generally, the activity of several enzymes involved in lignin biosynthesis increases under pathogen infection, following the accumulation of lignin. Ligni cation of cell walls limits the spread of enzymes and toxins of fungi to the host, and also limits the absorption of water and nutrients from the host. Many lignin biosynthesis-related proteins were down-regulated in our study ( Fig. 6A and Additional le 5: Table S3).. In particular, a number of peroxidases were down-regulated in response to CLas-infection, implicating that phenylpropanoid biosynthesis pathway might play signi cant roles on preventing CLas early infection and CLas might inhibit the formation of lignin by depressing the peroxidases to contribute to disease infection. Therefore, genes of many elicitors that can promote phenylpropanoid biosynthesis are potential targets for breeding CLas-tolerant cultivar.

Conclusions
In this study, a comparative proteome analysis of Citrus petiole protein pro les using TMT-labeled technology was carried out. A total of 777 DEPs were identi ed in response to CLas, which is signi cantly more than that identi ed in previous studies. We mainly showed 3 physiological and molecular processes that many DEPs involved. The data suggested that CLas infection could promote the carbohydrate metabolism (DEPs were generally significantly up-regulated), depress the photosystem (DEPs were significantly down-regulated) and activate/inhibit defense responses (some of DEPs were significantly up-regulated). Western blot analysis was carried out here to validate the reliability of proteome data with 4 representative DEPs of CsGBSS1, CsPSI, CsSAMT and CsLOX2.1. The data expand the CLas-mediated protein catalog in citrus plants. Identifying the early plant responses in response to CLas infection in petioles contributes to facilitating the researches on citrus resistance against CLas.

Plants materials
Two-year-old seedlings of sweet orange (Citrus sinensis cv. Newhall) were obtained from the Institute of Citrus Research located in Ganzhou, Jiangxi Province, China. They were grafted with bud sticks from qPCR-positive sweet orange plants. For mock-inoculated controls, the same types of plants were grafted with bud sticks from qPCR-negative sweet orange plants. All plants were grown under controlled conditions (natural photoperiod at 25 to 28°C) in an insect-proof greenhouse. Starting at 2 months after grafting, each plant was detected biweekly using qPCR for CLas as described [64]. The CLas in leaf petioles was rst observed at 3-4 months post grafting, and typical HLB symptoms appeared at 4-5 months post inoculation. One month after rstly observing CLas by qPCR was considered as the early infection stage. Three biological replicate samples of PCR-positive and PCR-negative plants were collected to extract total proteins for proteomic analysis. About 10-15 leaf petioles containing similar CLas quantity from 1-2 trees were sampled as a biological replicate.

Plant protein extraction, digestion and TMT-labeling
The petiole sample was ground in a mortar in the presence of liquid nitrogen. The cell powder was transferred to 50-ml centrifuge tubes, 10 ml of lysis buffer (8 M urea, 2% SDS, 1× Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland)) was added to each sample, followed by sonication on ice and centrifugation at 13,000 g for 15 min at 4℃. The supernatant was transferred to a fresh tube. For each sample, proteins were precipitated with ice-cold acetone at -20℃ overnight, the precipitations were cleaned with 50% ethanol and 50% acetone three times. 200 μg proteins was diluted by buffer (100 mM Tris, pH 8.0, 8 M urea) to 100 μl, then the solution was added 11 μl DTT (1 M) and incubated at 37 °C for 1 h. The treated samples were added into 10 kDa ultra ltration tube (Millipore, MA, USA) and centrifuged at 12,000 g for 10 min. Then 100 μl 55 mM iodoacetamide (IAA) was added to ultra ltration tube and incubated for 20 min protected from light at room temperature. After that, 50 mM triethylammonium bicarbonate (TEAB) was used as exchange buffer. Then proteins were tryptic digested with sequencegrade modi ed trypsin (Promega, WI, USA) overnight at 37 °C in a 1:50 trypsin-to-protein mass ratio, and the resultant peptide mixture was labeled using chemicals from the TMT reagent kit (Pierce Biotechnology, IL, USA). Proteins were labeled with the TMT as follows: TMT Sample1 was labeled with 131/129 (CLas/Mock), TMT Sample2 was labeled with 130/127, and TMT Sample3 was labeled with 128/131. Samples were then dried in vacuo.

High pH reverse phase separation
The peptide mixtures were redissovled in the buffer A (20 mM ammonium formate in water, pH 10.0, adjusted with ammonium hydroxide), and then fractionated by high pH separation using Ultimate 3000 system (ThermoFisher scienti c, MA, USA) connected to a reverse phase column (XBridge C18 column, 4.6 mm × 250 mm, 5 μm, (Waters Corporation, MA, USA). High pH separation was performed using a linear gradient. Starting from 5% to 45% buffer B (20 mM ammonium formate in 80% ACN, pH 10.0, adjusted with ammonium hydroxide) in 40 min. The column was re-equilibrated at initial conditions for 30℃. 12 fractions were collected, each fraction was dried in a vacuum concentrator for the next step.

Low pH NANO-HPLC-MS/MS analysis
The fractions were resuspended with 30 μl solvent buffer C (water with 0.1% formic acid) respectively, separated by nanoLC and analyzed by on-line electrospray tandem mass spectrometry. The experiments were carried out on an Easy-nLC 1000 system (Thermo Fisher Scienti c, MA, USA) connected to a Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scienti c, MA, USA) equipped with an online nano-electrospray ion source. 10 μl peptide sample was loaded onto the trap column (Thermo Scienti c Acclaim PepMap C18, 100 μm × 2cm), with a ow of 10 μl/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm × 15 cm) with a linear gradient, from 3% to 32% buffer D (ACN with 0.1% formic acid) in 120 min. The column was re-equilibrated at initial conditions for 10 min. The column ow rate was maintained at 300nL/min. The electrospray voltage of 2 kV versus the inlet of the mass spectrometer was used. The fusion mass spectrometer was operated in the data- Scaffold Q+ (version Scaffold_4.6.2, Proteome Software Inc., Portland, OR) was used to quantitate TMT Label Based Quantitation peptide and protein identi cations. Normalization was performed iteratively (across samples and spectra) on intensities, as described in Statistical Analysis of Relative Labeled Mass Spectrometry Data from Complex Samples Using ANOVA [65]. Medians were used for averaging. Spectra data were log-transformed, pruned of those matched to multiple proteins, and weighted by an adaptive intensity weighting algorithm. Differentially expressed proteins were determined by applying Mann-Whitney Test with unadjusted signi cance level p < 0.05 corrected by Benjamini-Hochberg.
Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Proteins were classi ed by GO annotation based on three categories: biological processes, cellular components and molecular functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) database was used to annotate protein pathways.
Quantitative real-time PCR Total RNA from citrus petioles was extracted using a plant RNA kit (Qiagen, Valencia, CA). Reverse transcription was carried out with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instruction. Total DNA from citrus petioles was extracted using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Ampli cation was performed in an ABI7500 Real-Time PCR system with SYBR Fast qPCR Mix (Takara, Dalian, China). Citrus GADPH gene was used as internal references to normalize the amount of RNA in different reactions. The relative mRNA quantities were calculated from the threshold cycle using the ΔΔCt method [66]. Each experiment was repeated three times. The details of the primers used in this experiment, please see (Additional le 7: Table S4).

Determination of the endogenous levels of salicylic acid and jasmonic acid
For the SA and JA content assay, CLas-inoculated and control citrus samples were collected in three replicates. Petiole tissues were collected, weighed and frozen in liquid nitrogen. For each sample, 0.1 g of the frozen tissue was extracted and quantitated for free SA and JA, as described previously [25,67]. In brief, the tissue was ground into powder and homogenized in 1 mL of methanol-H 2 O-acetic acid (80:19:1). After extraction overnight at 4℃ and centrifugation, the supernatant was re-extracted with the solution described previously. After the addition of 1 mL chloroform and further centrifugation, the organic phase containing the free SA was dried in a speed vacuum with heat (∼40℃). The residue was resuspended in 0.5 mL of methanol, ltered and analyzed by Ultra Performance Liquid Chromatography (UPLC). UPLC was performed on an ACQUITY UPLC@BEHC18 column (50 mm × 2.1 mm, 1.7 μm) run at 40℃ with a ow rate of 0.4 mL min −1 . The analytes were eluted from the column with a mixed solvent of water with 0.1% acetic acid (solvent A) and methanol with 0.1% acetic acid (solvent B) using a linear gradient mode. The ratio of A and B was 90:10 from 0 s to 3 min, and this ratio changed linearly from 90:10 to 10:90 between 3 and 4 min. The ratio of 90:10 was nally maintained from 4 to 7 min. The authenticity of the SA/JA from citrus petiole extract was veri ed based on the retention times and spectral properties, which matched perfectly to those of commercial SA/JA standards.
Cloning, expression and puri cation of proteins The coding sequence of targeted protein fragment was cloned into pET28a/pET32a (Novagen) and transformed into E. coli strain BL21(DE3) for expression. The refolding of the recombinant proteins expressed in inclusion bodies was carried out as previously described [68]. The recombinant proteins were puri ed via 6×His tag, and then detected by SDS-PAGE with the molecular mass marker (RTD6105, Beijing Real-Times Biotechnology Co. Ltd., China. The concentration of the puri ed protein was measured using the BCA TM Protein Assay Kit (TransGen Biotech), and the protein was then frozen at -80℃ in small aliquots until use.

Preparation of ployclonal antibody and Western blot analysis
The aim band was excised and used as antigens for antibody production. Antibodies were produced in rabbit by HuaAn Biotechnology Company. Citrus petioles were ground in liquid nitrogen and then added   (C) All identi ed proteins were shown by a Volcano Plot. Differentially expressed proteins were shown as red (up-regulation) and green (down-regulation) spots.