Post-transcriptional regulation of fruit ripening and disease resistance in tomato by the vacuolar protease SlVPE3
- Weihao Wang†1,
- Jianghua Cai†1, 2,
- Peiwen Wang1, 2,
- Shiping Tian1, 2 and
- Guozheng Qin1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 6 January 2017
Accepted: 22 February 2017
Published: 7 March 2017
Proteases represent one of the most abundant classes of enzymes in eukaryotes and are known to play key roles in many biological processes in plants. However, little is known about their functions in fruit ripening and disease resistance, which are unique to flowering plants and required for seed maturation and dispersal. Elucidating the genetic mechanisms of fruit ripening and disease resistance is an important goal given the biological and dietary significance of fruit.
Through expression profile analyses of genes encoding tomato (Solanum lycopersicum) cysteine proteases, we identify a number of genes whose expression increases during fruit ripening. RNA interference (RNAi)-mediated repression of SlVPE3, a vacuolar protease gene, results in alterations in fruit pigmentation, lycopene biosynthesis, and ethylene production, suggesting that SlVPE3 is necessary for normal fruit ripening. Surprisingly, the SlVPE3 RNAi fruit are more susceptible to the necrotrophic pathogen Botrytis cinerea. Quantitative proteomic analysis identified 314 proteins that differentially accumulate upon SlVPE3 silencing, including proteins associated with fruit ripening and disease resistance. To identify the direct SlVPE3 targets and mechanisms contributing to fungal pathogen resistance, we perform a screening of SlVPE3-interacting proteins using co-immunoprecipitation coupled with mass spectrometry. We show that SlVPE3 is required for the cleavage of the serine protease inhibitor KTI4, which contributes to resistance against the fungal pathogen B. cinerea.
Our findings contribute to elucidating gene regulatory networks and mechanisms that control fruit ripening and disease resistance responses.
KeywordsFruit ripening Disease resistance Protease Vacuolar processing enzyme RNA interference Quantitative proteome Protease inhibitor Tomato
Fruit are highly specialized plant organs that play a central role in seed maturation and dispersal in angiosperms. They are also valuable components of human diets, providing essential nutrients and a wide range of “bioactive” compounds that are important for human health . The ripening of fleshy fruit is a genetically programmed process that is associated with changes in color, texture, flavor, and susceptibility to pathogen infection [2, 3]. Substantial insights have been made into the mechanistic basis of ripening, including its regulation by transcription factors and the gaseous hormone ethylene, which plays a central role in the ripening of climacteric fruit [4, 5]. The ethylene biosynthetic and signal transduction pathway have been well characterized [6–8]. Recently, significant progress has been made in understanding the transcriptional control of fruit ripening using tomato (Solanum lycopersicum) as a model system. The transcription factors RIPENING-INHIBITOR (RIN) , NONRIPENING (NOR) , and COLORLESS NONRIPENING (CNR)  function as global regulators of ripening and act upstream of ethylene, while additional transcription factors that are required for normal ripening include TOMATO AGAMOUS-LIKE1 (TAGL1) [12, 13], HD-ZIP HOMEOBOX PROTEIN-1 (HB-1) , APETALA2a (AP2a) [15, 16], ETHYLENE RESPONSE FACTOR6 (ERF6) , ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE (APRR2-Like) , and FRUITFULL (TDR4/FUL1 and MBP7/FUL2) . In addition to transcriptional regulation, gene expression in fruit ripening can also be modulated by epigenetic or translational control [20–22]. However, unlike the relatively well studied and defined changes in the fruit transcriptome during ripening, little is known about its post-transcriptional regulation.
One mode of post-translational control involves proteases, enzymes that are widely distributed in all living organisms and that catalyze the hydrolysis of peptide bonds during or after translation . Proteases specifically cleave protein substrates either from the N- or C- terminus (aminopeptidases and carboxypeptidases, respectively) or within the molecule (endopeptidases) . Based on the active site residues or ions that carry out catalysis, proteases are generally grouped into five classes: aspartic, cysteine, metallo-, serine, and threonine proteases . Although initially considered to be purely degradative enzymes involved in intracellular protein turnover, it is now clear that they participate in the regulation of many critical physiological and cellular processes [23, 25]. The information housed in the MEROPS peptidase database  suggests that a plant genome sequence encodes hundreds of proteases belonging to dozens of unrelated families. These enzymes have been associated with a wide variety of biological processes, including programmed cell death (PCD), meiosis, seed coat formation, cuticle deposition, stomata development, flowering, pollen or embryo development, and chloroplast biogenesis . However, the molecular mechanisms of plant proteases in these processes, such as their substrates, remain elusive. Moreover, the functional roles of proteases in fruit ripening have not been well defined. Our previous study has shown that specific E2 ubiquitin-conjugating enzymes, the components of an ubiquitin-proteasome system, contribute to the ripening of tomato fruit . From this we infer that proteolysis is involved in the regulation of fruit ripening, but how proteases participate in this process and the nature of the underlying mechanisms are not known.
In the study presented here, we identified a number of cysteine protease genes whose expression increases during tomato fruit ripening. We then assessed the physiological importance for fruit ripening of one of these genes, SlVPE3, which encodes a vacuolar processing enzyme (VPE). VPEs are canonical cysteine proteases, harboring active cysteines in the catalytic site, and are responsible for the maturation or activation of specific vacuolar proteins in plants . Here, we show that suppression of SlVPE3 by RNA interference (RNAi) in tomato delays ripening-related traits, including lycopene accumulation and ethylene synthesis. Interestingly, the tomato fruit with reduced SlVPE3 expression exhibited increased susceptibility to the necrotrophic pathogen Botrytis cinerea, despite showing a reduced ripening phenotype. This was unexpected since unripe green fruit are typically less susceptible than ripe fruit to pathogen infections [3, 30]. To investigate how SlVPE3 affects these processes, we performed a comparative proteomic analysis using isobaric tags for relative and absolute quantification (iTRAQ). This suggested a large number of proteins as potential SlVPE3 targets, including some involved in fruit ripening and disease responses. We also carried out a screen for SlVPE3-interacting proteins to identify the direct targets of SlVPE3. We provide evidence that a Kunitz trypsin inhibitor 4, a serine protease inhibitor, functions downstream of SlVPE3 to regulate disease resistance in tomato fruit.
Expression of the protease SlVPE3 increases steadily during fruit ripening
According to the MEROPS protease database, the tomato genome has 14 VPE genes, five of which have previously been identified and named SlVPE1 to SlVPE5 . We named the other nine VPE genes SlVPE6 to SlVPE14 on the basis of their chromosomal location (Additional file 3: Table S3). All these VPE proteins are predicted to contain two conserved cysteine residues in the active sites (Additional file 4: Figure S1). Phylogenetic analysis revealed that tomato VPE proteins can be divided into several subgroups, with >50% bootstrap support (Fig. 1c), and high sequence similarity among the proteins was observed (Additional file 5: Table S4), suggesting gene duplications. We selected SlVPE3 for functional analysis because its expression was not only higher in fruit than in other organs, such as root, stem, and leaf, but also increased gradually during fruit ripening (Fig. 1d). SlVPE3 has been shown to be involved in controlling sugar accumulation , but its function in fruit ripening and the underlying molecular mechanisms are unclear.
SlVPE3 is required for tomato fruit ripening
The color change of ripening tomatoes from green to red is largely due to the degradation of chlorophyll and the accumulation of lycopene, which accounts for 70–90% of the total carotenoids in most tomato varieties [38, 39]. To establish the underlying causes of the color differences observed between wild-type and SlVPE3 RNAi ripe fruit, we measured the levels of lycopene. As shown in Fig. 2d, levels of lycopene in fruit from the SlVPE3 RNAi lines were <10% of wild-type levels at 38 dpa, suggesting that SlVPE3 expression affects lycopene accumulation during fruit ripening.
As with all climacteric fruit, those of tomato require an increase in ethylene biosynthesis to ripen . We investigated whether the delay in fruit ripening in the SlVPE3 RNAi lines correlated with ethylene production. It was observed that fruit of the repressed lines (3-4, 3-12, and 3-15) produced less ethylene than wild-type fruit at the same ripening stages (Fig. 2e). Ethylene regulates the expression of many genes associated with fruit ripening  and we observed that the expression of SlVPE3 increased during tomato fruit ripening (Fig. 1d). To determine whether the increased expression of SlVPE3 was ethylene-dependent, wild-type fruit at 35 dpa were treated with the ethylene precursor ethephon, which resulted in a substantial increase in expression (Fig. 2f). Taken together, these results suggest that ethylene induces the expression of SlVPE3, which in turn regulates ethylene synthesis in tomato fruit by a positive feedback loop.
Disease resistance is impaired in SlVPE3 silenced fruit
Quantitative proteome analysis identifies proteins that differentially accumulate upon SlVPE3 silencing
SlVPE3 modulates the accumulation of proteins associated with fruit ripening and disease resistance
SlVPE3 affects gene transcript levels indirectly
Screening for SlVPE3-interacting proteins
We also performed a yeast two-hybrid (Y2H) screen to identify proteins that interact with SlVPE3 using the Matchmaker Library Construction and Screening Kits (Clontech). SlVPE3 was used as bait against a tomato cDNA library constructed from wild-type fruit at the breaker stage (38 dpa). A total of 179 positive colonies were analyzed, leading to the identification of 49 proteins (Additional file 10: Table S9) involved in various cellular processes. Notably, several proteins associated with proteolysis were identified, including KTI4, which was then targeted for further analysis.
SlVPE3 protein interacts directly with KTI4 in vacuoles
To examine the intracellular localization of SlVPE3 and KTI4, their ORFs were separately introduced into a vector to generate a translational fusion with monomeric red fluorescent protein (mRFP) at the C-terminus. The constructs were separately introduced into Agrobacterium tumefaciens, which in turn was used to transform tobacco (Nicotiana benthamiana) leaves, from which mesophyll protoplasts were isolated. Protoplasts expressing mRFP alone served as a control. Confocal laser scanning microscopy showed that mRFP-tagged SlVPE3 (SlVPE3-mRFP) and KTI4 (KTI4-mRFP) both produced a strong signal in the vacuole, while the mRFP-only control displayed a fluorescent signal throughout the cell, but not from the vacuolar lumen (Fig. 9b). Notably, we were unable to detect the intracellular localization of SlVPE3 and KTI4 fused to green fluorescent protein (GFP) or mCherry, which might be due to degradation and/or protonation of GFP or mCherry under the acidic conditions that are found in vacuoles of higher plants .
To confirm the colocalization of SlVPE3 and KTI4, their ORFs were fused to mRFP and PRpHluorin (plant-solubility-modified ratiometric pH-sensitive mutants of green fluorescent protein), respectively, and the fusion proteins were co-expressed in tobacco leaves. PRpHluorin, derived from GFP, is a fluorescent pH sensor that has been successfully used to characterize the localization of vacuolar proteins . We observed that the fluorescent signals of mRFP co-localized with those of PRpHluorin, suggesting the subcelluar colocalization of SlVPE3 and KTI4 (Fig. 9c).
SlVPE3 participates in the cleavage of KTI4
To verify that SlVPE3 cleaves KTI4, the SlVPE3 and KTI4 ORFs were transiently expressed in tobacco (N. benthamiana) leaves. As shown in Fig. 10b, a band of the predicted molecular mass of the full-length KTI4 (~25 kDa) was generated by the anti-KTI4 antibodies, together with an additional faint band with a lower molecular mass. When SlVPE3 was co-expressed with KTI4 in tobacco, the abundance of the 25-kDa band decreased, concomitant with an increase in the intensity of the lower molecular mass band (Fig. 10b), suggesting that the 25-kDa band constitutes a precursor of the lower molecular mass band. Importantly, when KTI4 was co-expressed with a mutated form of SlVPE3 (SlVPE3C69G/C208G) in which conserved cysteine (C) residues (C69 and C208) from the active site were replaced by glycine (G), the changes in abundance of the two bands were abolished (Fig. 10b), indicating that SlVPE3 must be catalytically active for KTI4 to be cleaved.
We subsequently examined the cleavage of KTI4 by SlVPE3 in vitro. The predicted mature SlVPE3 polypeptide and KTI4 without the signal peptide were independently expressed in Escherichia coli as fusion proteins with a His-tag. Substantial amounts of recombinant SlVPE3 protein were obtained but did not show VPE activity (data not shown). We also expressed SlVPE3 in Saccharomyces cerevisiae BY4741 and Pichia pastoris GS115 cells, but did not detect proteolytically active recombinant proteins (data not shown). We next investigated the cleavage of the His-tagged recombinant KTI4 proteins (His-KTI4) in cell-free extracts of tobacco expressing intact or mutated SlVPE3 (SlVPE3C69G/C208G). A 25-kDa band, in addition to a band with lower molecular mass, was detected by the anti-KTI4 antibodies when extracts of tobacco expressing the empty plasmid were incubated with His-KTI4 (Fig. 10c). Two additional bands were observed in extracts of tobacco expressing intact SlVPE3, suggesting the cleavage of KTI4 by SlVPE3 (Fig. 10c). Gradual processing of KTI4 was observed when the reaction time was prolonged. The processing could be blocked in extracts of tobacco expressing the mutated form of SlVPE3 (SlVPE3C69G/C208G), suggesting specific cleavage of KTI4 by SlVPE3. To determine whether KTI4 is directly cleaved by SlVPE3, biotin-YVAD-fmk , a VPE inhibitor that efficiently inhibits the activity of SlVPE3 (Additional file 4: Figure S5), was added to the reaction. Application of the inhibitor blocked KTI4 processing (Fig. 10c), further demonstrating that KTI4 is a direct target of SlVPE3.
KTI4 does not inhibit the activity of SlVPE3
We also evaluated the effects of recombinant KTI4 proteins purified from E. coli on VPE activity. The recombinant KTI4 proteins exhibited activity against trypsin (Fig. 11b), indicating that they were active serine protease inhibitors. The incubation of the KTI4 proteins with extracts from tobacco leaves expressing intact or mutated SlVPE3 (SlVPE3C69G/C208G) did not significantly affect VPE activity, although this activity was inhibited by the specific inhibitor biotin-YVAD-fmk (Fig. 11c).
KTI4 contributes to resistance of fruit to pathogen infection
SlVPE3 is regulated by the RIN transcription factor
We also performed an electrophoretic mobility shift assay (EMSA) with the purified recombinant RIN protein. A band shift was observed when the purified RIN protein was mixed with the biotin-labeled probe (26-mer oligonucleotide) containing the CArG-box element. Binding of the RIN protein to the promoter fragment was out-competed by addition of an excessive amount of the corresponding unlabeled probe (competitor) containing an intact CArG box element, but not by the probe with a mutated CArG box element (Fig. 13c). This confirmed direct binding of RIN to the SlVPE3 promoter.
In recent years, substantial progress has been made in understanding the transcriptional control of fruit ripening, but relatively little is known about the regulation of fruit ripening at the post-transcriptional level. In the present study, we show that SlVPE3, a cysteine protease, is involved in fruit ripening. Repression of SlVPE3 resulted in a delay in ripening and made the fruit more susceptible to pathogen infection. Via a quantitative proteome study we found that SlVPE3 affects the abundance of a set of proteins associated with fruit ripening and disease resistance. Further analysis indicated that SlVPE3 participates in the cleavage of KTI4, a protease inhibitor that contributes to fruit disease resistance.
SlVPE3 regulates fruit ripening at the post-transcriptional level
Proteases represent one of the most abundant classes of enzymes in eukaryotes and reside in various cellular compartments, such as the cytosol, mitochondria, vacuoles, lysosomes, endoplasmic reticulum, or the extracellular domain [23, 27, 56, 57]. In A. thaliana, more than 800 proteases have been identified, representing approximately 2% of the genome . The distribution and the protease family size are well conserved within the plant kingdom . However, even though they have been demonstrated to participate in a variety of biological processes [23, 27, 59–61], their roles in fruit ripening remain unclear. In this study, we showed that SlVPE3, a gene encoding a vacuolar processing enzyme, is required for normal tomato fruit ripening.
The iTRAQ-based quantitative proteome analysis represents a powerful technique for high-throughput nonbiased discovery of protease targets [62–64], and has provided increased coverage and sensitivity for quantitative proteomics . Using this technique, more than 300 proteins that changed abundance upon SlVPE3 silencing were identified, of which a set was associated with fruit ripening. These results suggested that SlVPE3 regulates fruit ripening by affecting the accumulation of numerous ripening-related proteins. Notably, besides SlVPE3 targets, other proteins that changed abundance due to the delayed ripening were also identified. The proteins that differentially accumulated due to the activity of SlVPE3 itself might be identified by comparing the protein profiles between tomato leaves expressing an intact or mutagenized form of SlVPE3.
VPEs can degrade target proteins or activate substrate proteins from inactive precursor isoforms to active forms . Interestingly, a homolog of SlVPE3, SlVPE5 (formerly named LeCp), was reported to function as a transcription factor and to regulate the expression of ACS2 in tomato and tobacco (N. tabacum) leaves upon induction by the fungal elicitor EIX . LeCp was found to bind directly to the ACS2 promoter and it was proposed that LeCp functions as a protease in the cytoplasm and, upon elicitor signaling, enters the nucleus via binding by a small ubiquitin-related modifier (SUMO) protein, whereupon it acts as a transcription factor . In our study, we found that SlVPE3 affects the transcript levels of several ripening-related genes (Fig. 6). However, ChIP-quantitative PCR analysis indicated that SlVPE3 does not bind to the promoters of these genes. In addition, we found no evidence for direct binding of SlVPE3 to the ACS2 promoter. These results indicated that although LeCp, the homolog of SlVPE3, exhibits transcription factor activity that induces ACS2 expression in tomato leaves, SlVPE3 does not act as a transcription factor in tomato fruit during ripening. Accordingly, SlVPE3 may regulate the ripening-related genes at the post-transcriptional levels rather than at the transcriptional levels. Alternatively, SlVPE3 may modulate the expression of ripening-related genes indirectly through processing of specific transcription factors, which in turn regulate the expression of these genes.
Protease inhibitor KTI4 functions downstream of SlVPE3 to regulate fruit disease resistance
The susceptibility of tomato fruit to necrotrophic pathogens increases during fruit ripening [3, 30], and we hypothesized that since suppressing the expression of SlVPE3 resulted in a delay in fruit ripening, it might also affect fruit susceptibility to pathogen infection. It has also been reported that simultaneously suppressing the expression of genes encoding the cell wall-modifying proteins polygalacturonase (LePG) and expansin (LeExp1) reduces the susceptibility of ripening tomato fruit to B. cinerea infection . In this study, we found that a set of ripening-related proteins, including LePG and LeExp1, were less abundant in the SlVPE3 RNAi fruit than in wild type. Contrary to expectations, however, we observed that the SlVPE3 RNAi fruit were more susceptible to B. cinerea infection than the wild type (Fig. 3), suggesting that SlVPE3 may target proteins involved in defense responses, as well as those associated with ripening. Consistent with this idea, the proteomic data revealed that a number of proteins putatively associated with disease resistance have altered abundance in the SlVPE3 RNAi fruit. In A. thaliana, knocking out the expression of VPEγ was also demonstrated to increase the susceptibility of leaves to pathogens , but the underlying molecular mechanisms are not well understood.
To identify proteins directly responsible for the SlVPE3-regulated disease resistance, we screened for proteins that interact with SlVPE3 using quantitative affinity purification followed by mass spectrometry, which detects protein–protein interactions in the native cellular environment and is suitable for detecting weak and transient interactors. A protease inhibitor, KTI4, was identified as a SlVPE3-interacting protein and the interaction was confirmed by a Y2H assay (Figs. 8 and 9). Immunoblot analysis of fruit from wild-type tomato and SlVPE3 RNAi lines, and of tobacco leaves transiently expressing SlVPE3 and KTI4, using antibodies against KTI4 showed that SlVPE3 participates in the cleavage of KTI4 (Fig. 10a, b). Furthermore, KTI cleavage by SlVPE3 was confirmed by cell-free assays using recombinant KTI4 proteins (Fig. 10c). These data suggest that KTI4 is a direct target of SlVPE3.
KTIs are single-chain polypeptides of ~20 kDa belonging to the serine protease inhibitor family [45, 46]. A. thaliana KTI1 has been reported to be associated with leaf PCD in plant–pathogen interactions ; however, the function of KTI proteins in fruit disease resistance or their regulation has not been determined. In the present study, we found that KTI4 was processed by SlVPE3 (Fig. 10) and played a role in defense response of tomato fruit to B. cinerea (Fig. 12). Fruit in which KTI4 expression was suppressed by VIGS showed similar susceptibility to B. cinerea as those from the SlVPE3 RNAi lines (Figs. 3 and 12). Importantly, we observed that KTI4 does not inhibit SlVPE3 protease activity (Fig. 11). These data suggest that KTI4 functions as a downstream effector of SlVPE3 to regulate fruit disease resistance. Notably, suppression of KTI4 by VIGS did not affect fruit ripening (Additional file 4: Figure S6). The mechanism by which KTI4 affects pathogen infection remains unknown, but we speculate that it may target proteases that are secreted by fungal pathogens as virulence factors.
SlVPE3 is regulated at multiple levels
Due to the central roles of proteases in many biological processes and the irreversible nature of proteolysis, the action of proteases must be tightly controlled to prevent improper cleavage of substrate molecules . Protease activities are regulated at multiple levels. The regulation can occur at the transcriptional level, and at the protein level by activation of inactive zymogens, or by the binding of inhibitors and cofactors . Many proteases are synthesized as inactive zymogens, which contain inhibitory prodomains that must be removed for the protease to become active . The activation can be either autocatalytic or performed by other proteases. VPEs have been reported to be self-catalytically activated from inactive proproteins into active VPEs [51–53]. In contrast to the understanding of post-transcriptional regulation of VPEs activity, little is known about the regulation of VPEs at the transcriptional level.
An analysis of the SlVPE3 promoter region revealed two CArG box elements, which are binding sites for the RIN transcription factor, suggesting that SlVPE3 may be transcriptionally regulated by RIN. Gene expression analysis, combined with ChIP and EMSA assays, supported this hypothesis, and indicated that RIN regulates the expression of SlVPE3 by directly binding to its promoter (Fig. 13). RIN has been reported to directly regulate genes involved in a wide variety of biological processes, such as lycopene accumulation, ethylene production, chlorophyll degradation, and aroma formation [66, 67], but little is known about the regulation of protein degradation by RIN. We previously demonstrated that genes encoding E2 ubiquitin-conjugating enzymes, the components of the ubiquitin-proteasome, serves as direct targets of RIN , suggesting that RIN might regulate ubiquitin-mediated proteolysis. In this study, we show that RIN directly regulates protein degradation by targeting a specific protease, SlVPE3, which represents a previously unreported RIN direct target in a study involving ChIP coupled with DNA microarray analysis . Notably, the expression of SlVPE3 was only partially downregulated in the rin mutant fruit, suggesting that additional transcriptional regulators are necessary for SlVPE3 expression.
Our findings provide new insights into understanding the gene regulatory networks and proteolytic mechanisms that contribute to ripening and disease resistance in fruit. Future studies will investigate the target proteases of KTI4 and uncover the molecular mechanisms by which KTI4 regulates defense response in tomato fruit.
Seeds of wild-type tomato (Solanum lycopersicum cv. Ailsa Craig) and the ripening mutant rin in the cv. Ailsa Craig background were kindly provided by Dr. James J. Giovannoni (Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, USA). Plants were grown in a greenhouse under standard culture conditions, with a regular supply of fertilizer and supplementary lighting when required. Flowers were tagged at anthesis to accurately follow fruit ages through development. Fruit ripening stages used in wild type were mature green (MG), breaker (Br), orange (Or), and red ripe (RR), which were defined on basis of the color, size, shape, seed development, and the development of locular jelly in the fruit . These ripening stages (MG, Br, Or and RR) were on average 35, 38, 41, and 44 dpa, respectively. rin mutant fruit or transgenic lines were collected at the equivalent ripening stages, as determined by the number of dpa. Immediately after harvesting, pericarp tissue was collected, frozen in liquid nitrogen, and stored at −80 °C until use.
RNA isolation and quantitative RT-PCR analysis
RNA was isolated from the pericarp using the method of Moore et al. . The extracted RNA was treated with DNase I (Promega) and reverse transcribed using an oligo (dT)18 primer with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) to synthesize cDNA. Quantitative RT-PCR was carried out with the SYBR Green PCR Master Mix (Applied Biosystems) using the StepOne Plus Real-Time PCR System (Applied Biosystems). Gene-specific primers (Additional file 12: Table S11) were designed with the help of the Primer Express software 3.0 (Applied Biosystems). The following program was applied for PCR amplification in a volume of 20 μL: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Relative quantification of specific mRNA levels was performed using the cycle threshold (Ct) 2(−ΔCt) method . Expression values were normalized using ACTIN (SGN-U580609). Each experiment had three biological repeats, each with three technical replicates.
An alignment of the VPE protein sequences from tomato (S. lycopersicum) (Additional file 4: Supplementary text), rice (Oryza sativa), potato (Solanum tuberosum), tobacco (N. tabacum), pepper (Capsicum annuum), maize (Zea mays), soybean (Glycine max), citrus (C. sinensis), grape (Vitis vinifera) and A. thaliana was generated using ClustalX (version 2.1) software  with default multiple parameters and the PAM series protein weight matrix. The genedoc program was used to manually edit the alignment, which was then imported into MEGA (version 5.2) software  and the phylogenetic tree was constructed by the neighbor-joining statistical method using 1000 bootstrap replicates .
Construction of the RNAi vector and plant transformation
To construct the SlVPE3 RNAi plasmid, a 278-bp SlVPE3 fragment was amplified from cDNA of tomato fruit at 38 dpa with SlVPE3-specific primers (SlVPE3 RNAi/F: 5′-GTTCCCTCCACAGGGGTT-3′; SlVPE3 RNAi/R: 5′-AGATGAAAGAAAGTTTGTTCAGG-3′) and cloned into the pCR8/GW/TOPO Gateway entry vector (Invitrogen). The cloned fragment was subsequently transferred into the binary RNAi vector pK7GWIWG2D . The resulting construct was sequence confirmed and transformed into A. tumefaciens strain GV3101 , which was subsequently transformed into tomato (S. lycopersicum cv. Ailsa Craig) according to the method of Fillatti et al. . The presence of the transgene was verified by PCR in the T0 and T1 tomato generations.
Pericarp lycopene content was measured as described by Sun et al.  and expressed as mg kg−1 fresh weight. Each sample contained three replicates with five fruit per replicate and the experiment was repeated twice.
Ethylene production assay and ethephon treatment
To measure ethylene biosynthesis, fruit from transgenic T1 lines and the wild-type tomato were harvested at 38 and 41 dpa and placed in open jars for 3 h to avoid measuring “wound ethylene”, transiently synthesized as a consequence of picking. The jars were then sealed, incubated at room temperate for 2 h and then 1 mL gas samples were taken and analyzed by a gas chromatograph (SQ-206, Beijing, China) equipped with an activated alumina column and a flame ionization detector. Ethylene concentrations were calculated by comparing the peak length from the gas chromatograph with reagent grade ethylene standards of known concentration and normalizing for fruit weight. Each sample contained three replicates with five fruit per replicate and the experiment was repeated twice.
For the ethephon treatment, tomato fruit at 35 dpa were immersed for 10 min in a 50 mM fresh aqueous solution of ethephon (Sigma) or in water for the control. Pericarp samples were collected at various time intervals.
Protein extraction, iTRAQ labeling, and NanoLC–MS/MS analysis
Proteins were extracted from tomato fruit pericarp at 41 and 44 dpa as previously described . The isolated proteins were solubilized in protein buffer consisting of 500 mM triethylammonium bicarbonate (TEAB) and 1% SDS (w/v), pH 8.5, and the protein concentrations were determined by the Bradford method . One-hundred micrograms of protein from each sample were reduced, alkylated, and digested using the filter-aided sample preparation (FASP) method . The tryptic peptides were then labeled with the iTRAQ Reagents 4-plex Kit (Applied Biosystems) following the manufacturer’s protocol. Samples taken from wild-type and SlVPE3 RNAi fruit at 41 dpa were labeled with iTRAQ tags 114 and 115, respectively, while samples from wild-type and SlVPE3 RNAi fruit at 44 dpa were labeled with iTRAQ tags 116 and 117, respectively. The iTRAQ experiment was performed with two independent biological replicates. The iTRAQ-labeled samples were combined and subjected to high-pH reversed-phase chromatography. Briefly, the pooled iTRAQ-labeled peptides were reconstituted with buffer A (20 mM ammonium formate, pH 10, in water) and loaded onto a 4.6 × 250 mm, 150 Å size Durashell C18 (L) column containing 5 μm particles (Agela Technologies). The peptides were eluted at a flow rate of 0.8 mL min−1 using a gradient of 2% buffer B (20 mM ammonium formate in 80% acetonitrile, pH 10) for 5 min, 2–30% buffer B for 25 min, and 30–90% buffer B for 10 min. The system was then maintained in 90% buffer B for 10 min before equilibration with 2% buffer B for 10 min. The elution was monitored by measuring UV absorbance at 210 nm, and fractions were collected every 1 min. Forty-eight fractions were collected and pooled into a total of six fractions. After reconstitution in 0.1% formic acid, 8 μL of the combined iTRAQ-labeled peptides were submitted for NanoLC–MS/MS analysis.
The mass spectroscopy analysis was performed as previously described  using a NanoLC system (NanoLC–2D Ultra Plus, Eksigent) equipped with a Triple TOF 5600 Plus mass spectrometer (AB SCIEX). Protein identification and quantification for the iTRAQ experiments were performed using ProteinPilot™ 4.5 software (AB SCIEX). The mass spectra data were used to search the S. lycopersicum protein database ITAG2.4_proteins_full_desc.fasta, using the following parameters: (i) Sample type, iTRAQ 4-plex (Peptide Labeled); (ii) Cysteine alkylation, MMTS; (iii) Digestion, Trypsin; (iv) Instrument, TripleTOF 5600; (v) Species, None; (vi) Quantitate, Yes; (vii) Bias correction, Yes; (viii) Background correction, Yes; (ix) Search effort, Thorough; (x) FDR analysis, Yes. For iTRAQ quantification, the peptide for quantification was automatically selected using the Pro GroupTM algorithm (AB SCIEX) to calculate the reporter peak area. A reverse database search strategy  was used to estimate the global FDR for peptide identification. Only proteins identified below the 1% global FDR were ultimately exported for determining the meaningful cut-off value for the regulated proteins . Hierarchical clustering (Pearson algorithm) was carried out with PermutMatrix software .
Preparation of polyclonal antibodies
For SlVPE3-specific antibody preparation, a truncated form of SlVPE3 lacking the conserved domain was amplified from tomato cDNA using primers F (5′-AGGATATCGAAAGACACAACCTGCG-3′) and R (5′-ACGTCGACCTAACCAGCAGGGCGGAC-3′) and inserted into the pET-30a vector (Merck KGaA). The resulting plasmid was transformed into E. coli BL 21 (DE3) competent cells. For recombinant protein expression, E. coli was cultured overnight and then diluted 1:100 in Luria Broth medium. The bacteria were incubated at 37 °C until A600 reached approximately 0.5, then isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM to induce recombinant protein expression. The bacterial cells were incubated for an additional 3 h before the cells were collected by centrifugation for recombinant protein isolation. The recombinant protein was purified using Ni-NTA His Bind Resin according to the manufacturer’s manual (Merck KGaA), followed by further purification by 12% SDS-PAGE. The protein band corresponding to the predicted size of the recombinant SlVPE3 was excised from the gel and used to immunize rabbits at the Beijing Protein Institute Co., Ltd. Polyclonal antibodies that recognized SlVPE3 was affinity-purified from antisera using the AminoLink Plus Coupling Resin following the purification protocol (Thermo Scientific).
Polyclonal KTI4 antibodies were raised by injecting a rabbit with the synthetic peptide TYASVVDSDGNPVKAGAKYF followed by affinity-purification using the synthetic peptide.
Immunoprecipitation of SlVPE3-interacting proteins for SWATH-MS analysis
Proteins were isolated from 41 dpa tomato fruit using IP buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 50 μM MG132, 1 mM PMSF, and the protease inhibitor cocktail tablet (Roche). After centrifugation at 12,000 × g for 10 min, the supernatant containing the proteins was immunoprecipitated overnight at 4 °C with 50 μg of anti-SlVPE3 or pre-immune serum IgG (negative control) that was coupled to an agarose support, as described in the Pierce® Co-Immunoprecipitation (Co-IP) Kit (Pierce Biotechnology). The agarose beads were collected in spin columns and washed twice with IP buffer. The proteins were then eluted from the beads with 0.1 M glycine-HCl (pH 2.2), followed by reduction, alkylation, and digestion using the filter-aided sample preparation (FASP) method . The resulting peptides were collected, dried under vacuum, and redissolved in 0.1% formic acid for NanoLC-MS/MS analysis. Quantitative analysis of SlVPE3-interacting proteins was performed using the SWATH-MS method . Mass spectra were generated on a TripleTOF 5600 plus instrument (AB SCIEX) operating in the SWATH mode. Each sample contained three technical replicates and a t-test was used for statistical analysis. A P value <0.01 was considered to be significant.
Construction and two-hybrid screening of the tomato cDNA library was performed with the Matchmaker Library Construction and Screening Kit (Clontech). Total RNA was extracted from 38 dpa (Br stage) tomato fruit pericarp and mRNA was isolated and used for cDNA library construction. The tomato cDNA library constructed in the prey vector pGADT7 (AD) was screened with a SlVPE3 cDNA fragment encoding the mature protein cloned into the bait vector pGBKT7 (BD) in S. cerevisiae strain AH109 (Clontech), and positive clones were selected on SD/-Leu/-Trp/-His/-Ade medium supplemented with X-α-Gal.
To confirm the interactions between SlVPE3 and KTI4, the SlVPE3 cDNA fragment encoding the mature protein and the ORF of KTI4 were cloned into the AD and BD vectors, respectively, resulting in the SlVPE3-AD and KTI4-BD plasmids. Primers used for the vector construction are shown in Additional file 13: Table S12. The vectors were co-transformed into S. cerevisiae strain AH109 following the manufacturer’s manual (Clontech), and dripped on SD/-Leu/-Trp medium (SD/-2) and SD/-Leu/-Trp/-His/-Ade medium (SD/-4) containing X-α-Gal. As controls, AD and BD, KTI4-BD and AD, or SlVPE3-AD and BD were co-transformed. The experiments were repeated three times.
For subcellular localization analysis, the full-length SlVPE3 and KTI4 cDNAs were amplified by PCR (primer sequences in Additional file 13: Table S12) and individually cloned into the pCambia 2300-MCS-mRFP vector. The resulting plasmids were transformed into A. tumefaciens GV3101 , which was subsequently infiltrated into tobacco (N. benthamiana) leaves . For colocalization analysis, N. benthamiana plants co-expressing mRFP-tagged SlVPE3 (SlVPE3-mRFP) and PRpHluorin-tagged KTI4 (KTI4-PRpHluorin) under the control of the CaMV 35S promoter were generated. The plasmid containing PRpHluorin was kindly provided by Dr. Liwen Jiang (School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China). The tobacco plants were kept in the greenhouse for two days, and then mesophyll protoplasts were isolated  and observed under a Leica confocal microscope (Leica DMI600CS).
Protein cleavage assay
Proteins from the pericarp of wild-type and SlVPE3 RNAi fruit at 35 and 38 dpa were extracted using a phenol extraction method . The proteins were solubilized in lysis buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, and 1% dithiothreitol (DTT). Protein concentrations were determined by the Bradford method . The samples were separated by 12% SDS-PAGE and subjected to immunoblot analysis, as below.
For the KTI4 cleavage assay in tobacco (N. benthamiana), the full-length SlVPE3 and KTI4 were amplified using gene-specific primers (Additional file 13: Table S12) and cloned into the pCambia 2300 vector (Cambia) to generate the 35S:SlVPE3 and 35S:KTI4 constructs. A mutated form of SlVPE3 (SlVPE3C69G/C208G) was constructed by site-directed mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer’s instructions (primers are listed in Additional file 14: Table S13). The constructs were introduced into A. tumefaciens GV3101  and then expressed transiently in N. benthamiana leaves  as described above. After 2 days, total proteins were extracted with an extraction buffer containing 25 mM Tris · HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% (v/v) TritonX-100, 1 mM PMSF, and a protease inhibitor cocktail tablet (Roche). The homogenates were centrifuged at 12,000 × g for 10 min at 4 °C, and then the supernatants were subjected to immunoblot analysis, as below.
Cell-free assays for protein cleavage
The cell-free cleavage assay was carried out as previously described . The KTI4 coding sequence without the predicted signal peptide region was amplified from cDNA of tomato fruit using the primers KTI4-F (5′-GGGGTACCATGTCAACATTTTCTTCAGATCTT-3′) and KTI4-R (5′-GCGTCGACttaATCAGCCTTCTTGAAGTAA-3′) and cloned into the pCold I vector (Takara) to produce pCold I-KTI4. This construct allows an in-frame fusion of the coding region of KTI4 with an N-terminal His-tag. The plasmid was transformed into E. coli BL 21 (DE3) cells, and the recombinant protein expression and purification were performed as described above. For the cleavage assay, total proteins were extracted from N. benthamiana leaves that transiently expressed the intact or mutated form of SlVPE3 (SlVPE3C69G/C208G) using an extraction buffer containing 100 mM sodium acetate, pH 5.5, 1 mM PMSF, 100 mM DTT, and 100 μm E64-d. After two rounds of centrifugation at 12,000 × g for 10 min each at 4 °C, the supernatant was collected and the protein concentration was determined using the Bradford method . The total protein extracts (250 μg) were incubated with 200 ng of purified recombinant His-KTI4 in 250 μL extraction buffer for 0, 15, and 30 min at 20 °C. Wherever indicated, the VPE inhibitor biotin-YVAD-fmk was used at 100 μM by pre-incubation with the total protein extracts for 30 min before incubation with His-KTI4. The mixtures were subjected to SDS-PAGE followed by immunoblot analysis, as below.
For immunoblot analysis, proteins were separated by 12% SDS-PAGE and electrotransferred to an Immobilon-P PVDF membrane (Millipore). The membranes were blocked for 2 h at room temperature with 5% BSA in a PBS-Tween buffer (137 mM NaCl, 2.7 mM KCl, 8.1 mM NaH2PO4, 1.5 mM KH2PO4 and 0.1% Tween-20). Immunoblotting was conducted at 4 °C overnight with anti-KTI4 (1:1000) or anti-SlVPE3 (1:1000) antibodies. The membranes were washed with PBS-Tween (3 × 10 min), and then the secondary antibodies conjugated to horseradish peroxidase (Abmart) were added (1:5000). Immunoreactive bands were visualized using a chemiluminescence detection kit (SuperSignal®, Pierce Biotechnology). Equal loading was confirmed with an anti-actin antibody (Abmart).
VPE activity assay
VPE activity was measured as previously described  using a synthesized fluorescent VPE-specific substrate, Ac-ESEN-MCA [Acetyl-Glutamyl-Seryl-Glutamyl-Asparagine α-(4-Metyl-Coumaryl-7-Amide)] (Shanghai Bootech BioScience & Technology Co., Ltd). Proteins were extracted with a buffer containing 100 mM sodium acetate, pH 5.5, 1 mM PMSF, 100 mM DTT, and 100 μm E64-d from leaves of N. benthamiana that transiently expressed intact SlVPE3 (alone or co-expressed with KTI4) or the mutated form of SlPVE3 (SlVPE3C69G/C208G) for 2 days. The samples were centrifuged at 12,000 × g for 15 min and the supernatants were used for VPE activity assays. The crude enzyme extract was incubated with 100 μM Ac-ESEN-MCA in an acidic buffer containing 100 mM sodium-acetate (pH 5.5) and 100 mM DTT for 2 h at 20 °C. The fluorescence intensity was measured using a Synergy™ H4 Multimode Microplate Reader (Bio-Tek Instruments, Inc.) with an excitation wavelength at 380 nm and an emission wavelength at 460 nm. VPE inhibitor biotin-YVAD-fmk was added to a final concentration of 100 μM by pre-incubating with the crude enzyme for 30 min before Ac-ESEN-MCA addition, wherever indicated.
The trypsin inhibitor activity of recombinant KTI4 was determined as previously described . Trypsin (1 μg/μL, sigma) was pre-incubated with increasing concentrations of KTI4 in the assay buffer  for 30 min at 30 °C. The trypsin substrate N-benzoyl-L-arginine ethyl ester (Sigma) was then added to a final concentration of 0.25 mM and the changes in absorbance at 253 nm were monitored. Soybean trypsin inhibitor (Sigma) was used as a positive control. For the VPE activity assay with His-tagged recombinant KTI4 proteins, the purified KTI4 proteins (20 μg) were pre-incubated for 30 min with crude enzyme extracts from leaves of N. benthamiana that transiently expressed intact or the mutated form of SlPVE3 (SlVPE3C69G/C208G). Ac-ESEN-MCA was then added, as described above.
Virus-induced gene silencing
The VIGS assay was performed as previously described [87, 88]. The virus vectors pTRV1 and pTRV2 were provided by Dr. Daqi Fu (College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China). The specific KTI4 cDNA fragment was amplified using the primers F (5′-GGAATTCATGATGAAGAGCCTTGTTC-3′) and R (5′-CCGCTCGAGAGGACGTCCAGTGTTAAGTTCC-3′) and inserted into the pMD19-T vector (TaKaRa Bio). The resulting plasmid was transformed into E. coli and the sequence was verified. The cDNA fragment was subsequently cloned into the virus vector pTRV2 and then transferred to A. tumefaciens strain GV3101. For infiltration, equivalent aliquots of Agrobacterium strain GV3101 (with an optical density of 0.15 at 600 nm) containing pTRV1 or pTRV2 (empty or containing the insert) were mixed and injected into immature green stage tomato fruit. Agroinjected fruit were stored at room temperature (20 °C) for 14 days before infection with B. cinerea.
Infection of tomato fruit by B. cinerea was carried out as previously described . B. cinerea (1 × 105 conidia per ml) was applied to three sites that were equally spaced across the fruit surface. There were three replicates for each sample with at least ten fruit per replicate, and the experiment was repeated twice. Fungal growth was determined using quantitative PCR amplification of B. cinerea ACTIN 2 relative to the tomato ACTIN gene as described by Laluk and Mengiste . The following primer pairs were used for quantitative PCR: B. cinerea ACTIN 2 forward (5′-ACTCATATGTTGGAGATGAAGCGCA-3′), reverse (5′-AATGTTACCATACAAATCCTTACGGA-3′); tomato ACTIN forward (5′-ACAACTTTCCAACAAGGGAAGAT-3′) and reverse (5′-TGTATGTTGCTATTCAGGCTGTG-3′).
ChIP and EMSA
For the ChIP assay, pericarps of fruit at 41 dpa were excised, fixed in 1% formaldehyde under a vacuum for 20 min, then powdered in liquid nitrogen, and chromatin complexes were isolated and sonicated as previously described . The sonicated chromatin complexes were incubated with affinity purified polyclonal anti-SlVPE3/anti-RIN antibodies or pre-immune serum IgG (negative control) as previously described . The cross-linking was then reversed, and the amount of each precipitated DNA fragment was determined by real-time PCR using specific primers (Additional file 8: Table S7). Values are expressed as the percentage of DNA fragments that co-immunoprecipitated with specific (anti-RIN)  or non-specific (IgG) antibodies relative to the input DNA.
For the EMSA, recombinant His-tagged RIN protein was prepared as previously described , and purified using Ni-NTA His Bind Resin according to the manufacturer’s instructions (Merck KGaA). The ability of RIN to bind to biotin-labeled oligonucleotide probes was determined with the Lightshift Chemiluminescent EMSA kit (Thermo Scientific), as previously described .
Electrophoretic mobility shift assay
False discovery rate
Green fluorescent protein
Isobaric tags for relative and absolute quantification
Monomeric red fluorescent protein
Open reading frame
Programmed cell death
reverse transcription polymerase chain reaction
Sequential window acquisition of all theoretical mass spectra
virus-induced gene silencing
Vacuolar processing enzyme
We would like to thank Dr. Zhuang Lu for analysis of MS/MS and Dr. Daqi Fu from the College of Food Science and Nutritional Engineering, China Agricultural University for assistance with VIGS. We also thank the PRIDE team for the deposition of our mass spectrometry proteomics data to the ProteomeXchange Consortium. We thank PlantScribe (http://www.plantscribe.com/) for editing this manuscript.
This work was supported by the National Basic Research Program of China (973 Program; grant number 2013CB127103), the National Natural Science Foundation of China (NSFC; grant numbers 31172004), and the Youth Innovation Promotion Association CAS.
Availability of data and materials
The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride) under accession number PXD002980.
GQ conceived and designed the experiments. WW participated in quantitative RT-PCR analysis, RNAi vector construction, and plant transformation, ChIP assay, EMSA, iTRAQ analysis, and SlVPE3-interacting protein identification. JC carried out the Y2H assay, immunoblot analysis, enzyme activity assay, VIGS, and microbial inoculation. PW performed analyses related to lycopene production and ethylene biosynthesis. ST provided critical discussions. WW, JC, and GQ analyzed the data. GQ wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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