ABSTRACT
In tomato the Ve1-gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. Reciprocal grafts of resistant and susceptible tomato near-isolines were used to examine proteomic changes and, in particular, the effect of the Ve1-gene on the defence/stress protein cascade induced during Verticillium wilt. Based on label-free LC-MS, the results indicate that this defence response is cell-specific, correlates with overall fungal colonization and is mitigated by Ve1 function. The influence of the Ve1-gene in resistant tissues, however, is not actually transferred to susceptible tissues in the grafted plant.
Keywords: Verticillium wilt, tomato, proteome, defence genes, Ve R-locus, Ve1-gene
Although the hypersensitive reaction in foliar plant diseases has been described extensively,1,2 little is clear regarding plant defence strategies in vascular wilt diseases.3 Verticillium is a vascular fungus that affects more than two hundred economically important plants worldwide including alfalfa, cotton, cucurbits, eggplant, mint, olive, potato, sunflower, strawberry and tomato, with billions of dollars lost.4 In Verticillium wilt, the tomato Ve-resistance locus is thought to encode a receptor (NLR), which binds a pathogen effector setting off an immune cascade that curtails pathogen colonization.5 Breeding strategies have selected an inactivated Ve-R locus to improve yield and fruit quality but, unfortunately, also increased vulnerability to wilt. Currently susceptible stems often are grafted on resistant roots to provide, simultaneously, both an enhanced and resistant crop.6,7
Previous analyses of Craigella tomato near-isolines that are resistant (Ve1) or susceptible (ve1) to V. dahliae, race 1 (Vd1) indicated that during the compatible interaction plants develop a very heroic but unsuccessful systemic response involving many known plant defence/stress genes; surprisingly more modest changes occur during the incompatible interaction.8 Since a number of the highly elevated proteins are known to participate in a plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated defence response.
The Verticillium-derived effector AVE1 is believed to be perceived by the tomato Ve1 protein receptor9 but a direct interaction has not been demonstrated. In addition, the exaggerated defence response in the susceptible isoline appears not dependent on the Ve1-gene as the gene is truncated and not functional in a susceptible isoline.5 What is the actual target of Ve1 signalling and the mechanism of resistance both remain unknown.3 To better understand the contribution of genes to these processes (i.e. symptom development vs restricted fungal colonization) in grafted plants, we have undertaken genomic and proteomic studies of defence/stress gene expression in reciprocally grafted tomato with both susceptible (ve1) and resistant (Ve1) tissues. In a grafted plant, where the scion is susceptible, colonization is curtailed sufficiently to permit the enhanced crop production by the susceptible stem, leaves and flowers. It is unclear, however, if the induction of the defence cascade is cell-specific (i.e. determined by the genome of each individual cell) or transduced throughout the plant, a feature that could be relevant to future plant improvement. In the present study, we have used reciprocal grafts of susceptible (Cs) and resistant (Cr) near-isolines of the cultivar Craigella infected with Vd1 and proteomic analyses based on label-free LC-MS methods, to quantify protein changes in grafted plants and patterns of defence/stress protein induction.
Grafted plants were prepared from the Craigella isolines at the 4 leaf stage using the Japanese top grafting method10 with the cut just above the cotyledons. Healthy hybrids were inoculated by root dipping in gelatin solution containing 107 conidia/ml of Vd1.11 Infected plants were examined for disease symptoms and fungal levels or extracted for protein analyses at 10 days post infection (dpi) as previously described.8 As shown in Figure 1, both symptoms and Vd1 levels were consistent with typical wilt. Plants with a Cr rootstock were obviously taller and healthier while those with the Cs root were clearly shorter and wilted (upper panel). Equally the fungal biomass measurements (lower panel) indicated that fungal colonization also was typical of the root type. Both the lower and upper stems of plants with a Cs root contained significantly higher levels of fungal DNA than found in plants with a Cr root. As typically has been the case in Verticillium colonization experiments11 there was more than a twofold higher level in plants with a susceptible (Cs) root even in upper stems with a functional R locus. The Ve1-gene in the upper stem tissue did not protect the plant from wilt.
Figure 1.
Symptoms and fungal biomass in reciprocal grafts of resistant (Cr) and susceptible (Cs) tomato tissue infected with Verticillium dahliae. Infected, top-grafted plants were examined for symptoms (upper panel) and stems were severed at the soil line and cut into two parts at the graft. These upper and lower stem segments were extracted and assayed for fungal biomass (lower panel) at 10 dpi. Values are averages for the resistant (black) or susceptible (gray) stem tissue of 3 plants ± S.D.
For protein analyses, separate extracts were prepared from three plants with Cr roots or Cs roots. These were digested with trypsin for Nano-LC-MS/MS on a Thermo Fisher Orbitrap Fusion system with CID (collision-induced dissociation) and ETD (electron transfer dissociation) capabilities, coupled with a Dionex Ultimate 3000 nano LC with auto sampler.12 Progenesis QI for Proteomics (Waters, Milford, MA) software was used for data analyses; the mass spectra of peptides generated in the process were used to search the plant protein databases with MASCOT (Matrix Science, London, UK) to identify the proteins. High confidence protein identification was based on the identifications of at least two peptides, one of which was proteotypic (uniquely mapping to a single protein); quantification was based on 3 replicate extracts.
As illustrated in Table 1, analyses of the lower stem for each graft reflected previous comparisons between Vd1-infected Cr and Cs plants based on 2D gel protein fractionations.8 Of the 25 most elevated proteins in infected Cs lower stems relative to infected Cr plants, at least 15 clearly were defence/stress related; most were at least 2 fold higher in the Cs stem. When the same comparison was made between the upper stems (Table 2), the conclusion remained similar with the most elevated proteins being defence/stress related and in the Cs derived tissue, irrespective of the reciprocal lower stem/root genotype. Again, at least 15 were in the defence/stress category and 1.5–3 fold higher in the Cs tissue, irrespective of the opposite root type. Clearly, signals that result in the immune cascade do not significantly influence other tissues. Furthermore, as reported in previous studies, the fungal biomass appears not to be controlled by the level of defence/stress gene induction, which again raises the possibility that at least some of the disease symptoms are the result of the exaggerated cascade. Some of the elevated defence proteins may be enzymes that, in excess, cause damage to plant cell walls and actually facilitate fungal growth4,13 or may result in synthetic activities, such as giving rise to vascular coatings that contain fungal colonization (Robb et al. 1989) but also restrict water flow and cause wilt (Robb et al. 1983). The fact that defence/stress protein levels are reduced in tissue with functional Ve1 protein, suggests that the Ve1 receptor controls the response to limit the detrimental effects, a possibility that clearly needs to be examined.
Table 1.
Vd1 infected lower stem proteins.
| Average Normalised Abundances |
||||
| Accession |
Description |
Cs Lower Stem/Cs Root |
Cr Lower Stem/Cr Root |
Fold |
| gi|76363947 | pathogenesis-related protein [Solanum lycopersicum] | 8.95E+06 | 1.77E+06 | 5.07 |
| gi|460388877 | PREDICTED: acidic endochitinase-like [Solanum lycopersicum] | 1.07E+07 | 2.87E+06 | 3.73 |
| gi|350534760 | glucan endo-1,3-beta-D-glucosidase precursor [Solanum lycopersicum] | 1.50E+07 | 4.07E+06 | 3.7 |
| gi|224802 | protein p14,pathogenesis related | 6.18E+07 | 1.96E+07 | 3.15 |
| gi|350537435 | glucan endo-1,3-beta-glucosidase B precursor [Solanum lycopersicum] | 7.51E+07 | 2.39E+07 | 3.14 |
| gi|460388347 | PREDICTED: uncharacterized protein LOC101250018 [Solanum lycopersicum] | 6.09E+06 | 1.87E+07 | 3.08 |
| gi|58578270 | anionic peroxidase [Capsicum chinense] | 7.87E+05 | 2.03E+06 | 2.57 |
| gi|460384249 | PREDICTED: peroxidase 12-like [Solanum lycopersicum] | 2.01E+07 | 8.36E+06 | 2.4 |
| gi|88683140 | putative glucan endo-1,3-beta-D-glucosidase [Solanum tuberosum] | 3.54E+06 | 1.55E+06 | 2.28 |
| gi|111073733 | hypothetical protein [Nicotiana benthamiana] | 6.90E+05 | 3.02E+05 | 2.28 |
| gi|11321164 | beta-1,3-glucanase-like protein [Capsicum annuum] | 1.30E+07 | 5.72E+06 | 2.27 |
| gi|2114048 | water channel protein [Nicotiana excelsior] | 1.42E+06 | 3.18E+06 | 2.24 |
| gi|767827 | endochitinase, partial [Solanum chilense] | 1.91E+06 | 8.88E+05 | 2.15 |
| gi|168053476 | predicted protein [Physcomitrella patens] | 1.51E+05 | 7.24E+04 | 2.08 |
| gi|460404654 | PREDICTED: pathogenesis-related protein STH-2-like [Solanum lycopersicum] | 4.80E+07 | 2.32E+07 | 2.07 |
| gi|2921512 | GF14 protein [Fritillaria agrestis] | 1.98E+07 | 1.01E+07 | 1.96 |
| gi|565380238 | PR10 protein [Solanum lycopersicum] | 2.46E+06 | 1.28E+06 | 1.92 |
| gi|3413481 | serine protease [Solanum lycopersicum] | 1.93E+06 | 1.03E+06 | 1.88 |
Filters: Anova (p) < 0.05, Peptides > 1, 25 Highest Fold Values
Table 2.
Vd1 infected upper stem proteins.
| Average Normalised Abundances |
||||
|---|---|---|---|---|
| Accession | Description | Cr Upper Stem /Cs Root | Cs Upper Stem /Cr Root | Fold |
| gi|2114048 | water channel protein [Nicotiana excelsior] | 6.40E+06 | 1.25E+06 | 5.13 |
| gi|460400217 | PREDICTED: thaumatin-like protein-like [Solanum lycopersicum] | 2.70E+06 | 9.50E+05 | 2.84 |
| gi|350538633 | TSI-1 protein [Solanum lycopersicum] | 3.16E+06 | 8.39E+06 | 2.66 |
| gi|170458 | threonine deaminase, partial [Solanum lycopersicum] | 6.88E+06 | 1.43E+07 | 2.08 |
| gi|350538487 | formate dehydrogenase [Solanum lycopersicum] | 7.02E+05 | 1.46E+06 | 2.08 |
| gi|565380238 | PR10 protein [Solanum lycopersicum] | 9.51E+05 | 1.77E+06 | 1.86 |
| gi|565386495 | PREDICTED: peroxidase 12-like [Solanum tuberosum] | 2.55E+06 | 4.50E+06 | 1.77 |
| gi|565347758 | PREDICTED: pathogenesis-related protein STH-2-like [Solanum tuberosum] | 1.34E+07 | 2.33E+07 | 1.75 |
| gi|157849720 | heat shock protein 81–4 [Brassica rapa] | 4.92E+05 | 8.54E+05 | 1.74 |
| gi|460384249 | PREDICTED: peroxidase 12-like [Solanum lycopersicum] | 3.78E+06 | 6.44E+06 | 1.71 |
| gi|372995481 | pathogenesis-related protein PR10 [Nicotiana tabacum] | 2.54E+06 | 4.35E+06 | 1.71 |
| gi|76363947 | pathogenesis-related protein [Solanum lycopersicum] | 2.68E+06 | 4.54E+06 | 1.7 |
| gi|767827 | endochitinase, partial [Solanum chilense] | 1.29E+06 | 2.18E+06 | 1.69 |
| gi|568786980 | pathogenesis-related protein STH-2-like [Solanum lycopersicum] | 1.46E+07 | 2.42E+07 | 1.66 |
| gi|460404654 | PREDICTED: pathogenesis-related protein STH-2-like [Solanum lycopersicum] | 1.77E+07 | 2.92E+07 | 1.65 |
| gi|460395782 | PREDICTED: peroxidase 3-like [Solanum lycopersicum] | 4.89E+05 | 7.76E+05 | 1.59 |
| gi|350538783 | aquaporin-like [Solanum lycopersicum] | 4.19E+06 | 2.67E+06 | 1.57 |
| gi|565352690 | PREDICTED: suberization-associated anionic peroxidase 2-like [Solanum tuberosum] | 7.49E+05 | 1.15E+06 | 1.54 |
| gi|224802 | protein p14,pathogenesis related | 2.11E+07 | 3.24E+07 | 1.53 |
Filters: Anova (p) < 0.05, Peptides > 1, 25 Highest Fold Values
Funding Statement
This study was supported by NSERC (R.N.N. and J.R.) and NIH, NHLBI (A.K. and NSERC (R.N.N. and J.R.)); National Heart, Lung, and Blood Institute [N01-HV-00245]; Natural Sciences and Engineering Research Council of Canada [6768-2009 RGPIN]; Natural Sciences and Engineering Research Council of Canada [2961-2010 RGPIN];
Acknowledgments
We thank Drs. X. Luo and K.V. Soman (UTMB Proteomics Center) for help with the peptide mass spectrometry and proteomic analyses.
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