Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato

Abstract

The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during compatible or incompatible interactions. A new member of the PR-5 family was identified that accumulated early in both types of interaction. Other pathogenesis-related proteins appeared in compatible interactions only, concomitantly with disease development. This study demonstrates the feasibility of using proteomics for the identification of known and novel proteins in xylem sap, and provides insights into plant-pathogen interactions in vascular wilt diseases.

Figure 1

F. oxysporum infection causes accumulation of disease-related proteins in tomato xylem sap. Five-week-old GCR161 plants were either mock-inoculated (C) or inoculated with the compatible race 2 isolate Fol007 (Fol). After 3 weeks, when F. oxysporum-inoculated plants showed severe disease symptoms, sap was collected from individual plants, concentrated and analyzed with SDS-PAGE on a Tris-Tricine gel. Proteins were visualized by silver staining. Lanes represent sap from different plants. Molecular masses of marker proteins are indicated on the left (in kD). The most abundant disease-related proteins are indicated on the right, designated according to their estimated sizes.

Figure 2

Time-dependent accumulation of disease-related proteins in compatible and incompatible interactions. GCR161 plants were mock-inoculated (Control) or inoculated with the incompatible race 1 isolate Fol004, the compatible race 2 isolate Fol007, or the compatible race 3 isolate Fol029. Sap was collected at 1, 2, or 3 weeks after inoculation and analyzed as described in Figure 1. The disease-related proteins p12, p15, p22, p34, and p35 are indicated, as is the p10 protein present in healthy plants.

Figure 3

Most disease-related proteins bind to negatively charged Sepharose. After pH adjustment, xylem sap of tomato collected 3 weeks after infection with a compatible race of F. oxysporum (Fol007) was incubated with either SP- or Q-Sepharose with affinity for basic or acidic proteins, respectively. Proteins were washed off the Sepharose beads with 250 mm NaCl. Proteins were separated in Tris-Tricine gels and silver-stained. T, Total sap protein; NB, non-bound fraction; B, bound fraction.

Figure 4

Evidence for Asn deamidation. Shown are two examples of peptides whose molar mass distribution corresponds to partial deamidation of Asn residues. A, This cluster of peaks in a MALDI-TOF spectrum of p35 corresponds to the tryptic peptide NGNGLPSPADVVALCNR (predicted m/z of ¹²C monoisotopic peptide: 1,753.86 D) with partial deamidation of Asn residues in both Asn-Gly pairs. At this peptide molar mass size, the ¹²C monoisotopic peptide peak (MH⁺) should be at least equal to the one with one ¹³C atom (MH⁺ + 1), as shown in B. The higher abundance of the MH⁺ + 1 and MH⁺ + 2 peaks and the presence of two Asn-Gly pairs in the peptide suggests deamidation of Asn leading to 1 D mass increase per Asn. B, Predicted isotope distributions of the peptide described in A (lower trace) and with one (middle trace) or two (upper trace) Asn residues deamidated. Chemical formulae used for the calculations are shown next to the traces. From this peak shape, it was concluded that one and in some cases two Asn residues were deamidated. C, In this MALDI-TOF spectrum of p22, the cluster of peaks at the left corresponds to TNCNFNGAGR of PR-5x (predicted m/z of ¹²C monoisotopic peptide: 1,110.48 D). At this peptide molar mass size, the ¹²C monoisotopic peptide peak (MH⁺) should be higher than the one with one ¹³C atom (MH⁺ + 1), as seen for the larger peptide on the right (predicted m/z: 1,141.61). The higher abundance of the MH⁺ + 1 peak and the presence of an Asn-Gly pair in the peptide strongly suggests deamidation of Asn.

Figure 5

The p22 band contains a novel PR-5 protein. The vacuolar PR-5 proteins NP24 (accession no. P12670) and AP24 (the full translation product of The Institute for Genomic Research [TIGR] tentative consensus sequence TC52651 is shown) are aligned with the putative translation product (PR-5x) of the newly identified cDNA. Asterisks below the alignment indicate divergence in sequence between the proteins. White triangles above the aligned sequences indicate carboxyl-terminal amino acids of peptides predicted for a trypsin digest of any of the proteins in the alignment. Predicted tryptic peptides of PR-5x are numbered (T1–17). Peptides corresponding to MS/MS sequences are in bold. Peptides whose mass corresponds to peaks in the peptide mass fingerprint are underlined (including peptides T3–5 and T4–5 with missed cleavages; see Table IV). Cleavable N- and C-terminal signal sequences are in lowercase. Mature N termini were confirmed experimentally for NP24 (King et al., 1988) and AP24 (=P23; Rodrigo et al., 1991; Woloshuk et al., 1991); mature C termini are predicted by comparison with tobacco AP24 (Melchers et al., 1993).

Figure 6

The coding sequence of PR-5x is highly similar to coding sequences of vacuolar PR-5 proteins. PR-5x encoding cDNA (GenBank accession no. AY093595) is aligned with coding sequences for the vacuolar PR-5 proteins NP24 (accession no. AF093743; Jia et al., 2000) and AP24 (Ruiz-Medrano et al., 1992; Rodrigo et al., 1993); the sequence shown here is TC52651 from the TIGR Lycopersicon Gene Index, which includes the start codon. The line above PR-5x cDNA between arrow heads (≫–≪) indicates the sequence of a tomato-F. oxysporum-interaction-specific cDNA-AFLP fragment. Stop codons and potential start codons are bold and underlined. Coding sequences are in uppercase. Poly(A) addition sites, based on the 3′ ends of PR-5x cDNA clones, are indicated (✓), with numbers corresponding to the number of cDNAs ending there. Underlined sequences in PR-5x correspond to forward and reverse primers used for cloning of overlapping fragments of the PR-5x cDNA.

Figure 7

PR-5x belongs to a subgroup of PR-5 proteins that diversified in Solanaceae. All available full-length (predicted) protein sequences of PR-5 proteins of Arabidopsis (At), tomato (Le), tobacco (Nt), and potato (St), were aligned. Additions to the core PR-5 consensus sequence at N termini (signal sequences) and C termini (vacuolar targeting sequences or other extensions) were trimmed. This alignment was used to construct a phylogenetic tree. Only the clade containing PR-5x is shown. Published sequences are referred to by protein names: NP24 (accession no. P12670) and AP24 (accession no. CAA50059; complete sequence derived from TC52651 of the TIGR Lycopersicon Gene Index) from tomato; OSML13 (accession no. P50701), OSML35 (accession no. P50703), and OSML81 (accession no. P50702) from potato; AP24 (“osmotin”, accession no. P14170), PR-5d (accession no. BAA11180), PR-R1 (“major isoform”, accession no. P13046), and PR-R2 (“minor isoform”, accession no. P07052) from tobacco and AtOSM34 (accession no. CAA61411) from Arabidopsis. Remaining sequences are either derived from tentative consensus (TC) sequences in TIGR databases (Quackenbush et al., 2001) or database accessions. All sequence names are preceded by species abbreviations. Bootstrap percentages are provided for branches receiving 70% or more support. Branch length reflects the extent of sequence divergence. Thick arrows indicate secreted isoforms. All other proteins are known or predicted to be vacuolar (based on C-terminal propeptides). Only the two PR-R isoforms of tobacco are acidic.

Figure 8

Immunodetection of PR-2 and PR-3 isoforms in tomato xylem sap. Five-week-old C32 plants were either mock-inoculated (C) or inoculated with the compatible race 2 isolate Fol007 (Fol). Three weeks after infection, xylem sap proteins were isolated, separated in a Tris-Tricine gel, and blotted for immunodetection with antibodies raised against tobacco chitinase (Chi) or glucanase (Glu). Molecular masses (in kD) of marker proteins are indicated on the left.