Expression and purification of Xyn11A in Pichia pastoris
The yeast Pichia pastoris was transformed with the xyn11A cDNA under the control of the AOX1 promoter to induce the production of Xyn11A by methanol and its secretion by making use of its own signal peptide. Yeast transformant PICXYN18 showed abundant xylanase secretion in plates and in liquid culture and was selected for all subsequent experiments (Fig. 1A and 1B). Supernatant from a methanol-induced culture of this transformant showed two new polypeptides having masses around that predicted for the mature Xyn11A [10], 20.6 kDa. These expression products were then purified from the culture medium by a two-step protocol consisting of differential ammonium sulphate precipitation and gel exclusion chromatography. Three chromatographic fractions showed high xylanase activity and the presence of just the two new polypeptides by SDS-PAGE (Fig. 1C). These three fractions were pooled and dialyzed against water overnight and the resulting purified Xyn11A showed a protein concentration of 46 μg/ml and a specific activity of 122.7 U/mg protein. In order to check if the two protein bands observed in the purified xylanase fraction were both the product of the xyn11A gene, the two protein bands were cut from the gel and were subjected to peptide mass fingerprinting at the proteomic facility of the Centro Nacional de Biotecnología http://proteo.cnb.csic.es. Both bands were identified as the same protein, Xyn11A. Moreover, we used SELDI-TOF mass spectrometry to analyze the purified xylanase fraction and, surprisingly, the heterogeneity of the purified xylanase was higher than expected (Fig. 1D), with at least 7 different species differing slightly in mass. The reason for this phenomenon may be differences introduced by the Pichia glycosylation system from molecule to molecule [13, 14], or alternatively an incomplete processing by Pichia of the putative propeptide in the protein, as has been observed before for other proteins [15, 16].
The purified enzyme was characterized and the kinetic parameters were determined. We estimated both the optimal temperature, by carrying out the enzymatic reactions at different temperatures, and the thermal stability of Xyn11A, by assaying residual activity after incubation at different temperatures for 1, 2, 4, 10 or 15 minutes. The optimal temperature was about 45°C, but the enzyme lost activity rapidly above 35°C (not shown). Since the activity measured at 40 or 45°C seems to be the product of a decreasing quantity of a very active enzyme, the assay temperature chosen for all future incubations was 35°C. Concerning the effect of the pH, Xyn11A showed an optimal activity at approximately pH 5.0, in contrast to the extremely high predicted pI for the mature enzyme of 9.1 [10], but in accordance with the usually moderately acid pH of the B. cinerea extracellular medium [17], and the enzyme was very stable from pH 3 to 7 up to 4 hours , at room temperature (not shown). By using the optimized assay conditions, we calculated the Km of the enzyme for the substrate beechwood xylan resulting in a value, 7.1 g/l, in the same range as what has been found for other fungal xylanases [18].
Xyn11A has necrotizing activity on tomato and tobacco leaves
In order to check if, as previously hypothesised, Xyn11A was able to induce necrosis on plants, the purified enzyme dissolved in water at a concentration of 70 μg/ml was infiltrated in tomato and tobacco leaves and its effect was recorded for several days (Fig. 2A and 2B). The area of the leaf treated with Xyn11A became necrotic about 2-5 days after infiltration, but this effect was absent in areas infiltrated with water or with the control protein Bovine Serum albumin (not shown). Positive controls made using the commercially available xylanase EIX from T. viride [12] caused similar effects as those caused by Xyn11A (Fig. 2C). Both the appearance of the lesions as well as its time course were similar to what has been previously reported for the xylanases from T. viride and T. reesei [11, 19, 20].
The ability of Xyn11A to induce the production of reactive oxygen species (ROS) in the infiltrated leaves was also studied by determining the production of hydrogen peroxide with diaminobenzidine (DAB), since H2O2 is one of the landmarks of the hypersensitive response [5]. Leaves were first infiltrated with purified Xyn11A as before, then treated with DAB as explained in materials and methods, and finally, decolorized with ethanol to allow easier visualization of the dark, reduced DAB precipitate (Fig. 2D). A clear brown precipitate was observed only in the leaf areas that had been infiltrated with Xyn11A but not in those not infiltrated or infiltrated with water. Positive controls were made by infiltration with T. viride EIX xylanase (not shown) and by wounding the tip of leaves, which has been shown previously to induce the production of ROS [21], and the response obtained in both cases was similar to that obtained for Xyn11A.
The ability of Xyn11A to induce necrosis was cultivar dependent in the case of tobacco. Three tobacco cultivars differing in their susceptibility to B. cinerea were assayed for their capacity to develop necrosis after infiltration with Xyn11A, Nicotiana tabacum cv. Havana, and two local varieties, Alcalá and Paraíso. The diameter of infection areas for the two local varieties were about half the value obtained for Havana, and similarly, Xyn11A induced a response when infiltrated in the leaves of the Havana cultivar that was much stronger than that obtained for the other two (Fig. 3).
Necrotizing activity of Xyn11A is independent of its xylanase activity
One obvious question at this point was if the necrosis inducing activity of Xyn11A and its enzymatic activity on xylan were independent properties of the enzyme, what would rule out the possibility that the actual inducers of necrosis were xylan oligomers. This has already been proven for the necrosis inducing xylanases from T. reesei [11] and T. viride [22], and in the latter case the eliciting epitope has been mapped to a region of the enzyme surface that is away from the catalytic site [23]. In order to check if this is also true for Xyn11A, we generated four different mutants of Xyn11A in which either one of the two glutamic acid residues in the active site that are essential for the xylanase activity was substituted by either Gln or Ser. The four mutant proteins were expressed in P. pastoris and purified as explained before for the wild-type Xyn11A. All four proteins were unable to degrade xylan (not shown), but retained the same necrotizing activity as the wild-type, as well as the ability to induce the production of H2O2 (Fig. 4). These results confirm that in order to induce the development of necrotic lesions in plant tissues, Xyn11A does not need to be able to hydrolyze xylan.
The xylanase activity of Xyn11A is not necessary to complement the xyn11A mutant phenotype in B. cinerea
The lack of the protein Xyn11A in B. cinerea makes the fungus less virulent than the wild-type [10]. Due to the fact that the two xyn11A mutants already available [10] showed a somehow variable phenotype with respect to virulence, we generated 6 new xyn11A mutants by transforming the wild-type strain B05.10 with the same construction used before [10]. All of the new mutants were characterized by Southern-blot and PCR as having a single integration of the foreign DNA at the xyn11A locus, similarly to the previous ones [10]. The virulence was assayed for these 6 new mutants and it was shown again that the deletion of xyn11A resulted in a decrease in virulence. Fig. 5 shows the reduction of infectivity for one of these new mutants, N23, which was used for the rest of the work. As discussed above, one of the hypothesis that could explain this effect is a contribution of Xyn11A to induce death of the plant cells surrounding the infected area. If this is true, and taking into account that the necrotizing and the xylanase activities are independent, then the point-mutated xyn11A genes coding for proteins with no xylanase activity should be able to complement the xyn11A mutation in B. cinerea, reverting the phenotype back to full virulence. In order to check if this is the case, three plasmids were generated (pNRXYN, pNRX122S and pNRX214S), all containing the nourseothricin resistance cassette along with the whole xyn11A gene, including the 5' and 3' untranslated regions, in three variants: the wild-type gene or an altered gene coding for one of the two site-directed mutant proteins described above, E122S and E214S. The three plasmids were transformed into the xyn11A mutant N23 and hygromycin and nourseothricin-resistant transformants were purified by single conidia isolation and checked by PCR for the presence of the transforming xyn11A gene. The oligonucleotides used were TX-Sal (5'-ACCAAGCAAGATACCAAAGTC-3') and MUT-X-XY (5'-AATCCGCGAGTCTGGATC-3') and amplified a 2.3-kb region containing the whole xyn11A ORF plus 1 kb and 0.5 kb of the 5' and 3' untranslated regions, respectively. This fragment can arise only from the foreign transforming DNA since the original xyn11A copy had been interrupted by a 2.7-kb hygromycin resistance cassette. A second PCR was made to corroborate the persistence of this interrupted xyn11A copy already present in the N23 mutant. This time the oligonucleotides used were MUT-X-H (5'-TCGATGCGACGCAATC-3') and MUT-X-XY (5'-AATCCGCGAGTCTGGATC-3'), which bind, respectively, to the hygromycin resistance cassette and to the xyn11A gene. This PCR would generate a 1.7-kb fragment only if the original xyn11A locus is still interrupted with the hygromycin resistance cassette. It was done to rule out a double recombination at the xyn11A locus that may generate a wild-type xyn11A gene from the copy interrupted by the hygromycin cassette and the transforming copy with the site-directed mutation. 3 to 4 transformants were identified for the three transformations that fulfilled these requirements and all of them were assayed for their virulence on tomato leaves. Representative results are shown in Fig. 5. Although with differences among individual transformants, all of them were more virulent than the xyn11A mutant N23 and close to the wild-type strain B05.10. These results clearly indicate that Xyn11A is contributing to virulence with its necrotizing activity and not with its xylanase activity, since the two mutant proteins had been previously shown to be unable to degrade xylan, but to retain the necrotizing activity when expressed in P. pastoris (Fig. 4).
The contribution to virulence of the non-xylan-degrading Xyn11A proteins was also assayed in a different way, by exogenously providing the pure proteins to the infection process. Firstly, the wild-type Xyn11A protein and the mutant protein E214S were infiltrated in tomato leaves. Four hours later, the leaves were cut and the infiltrated areas were infected with the wild-type B. cinerea strain B05.10 or the xyn11A mutant strain N23. The presence of the Xyn11A protein, with or without xylanase activity, enhanced considerably the progression of the infection for both the wild-type and the xyn11A mutant strains (Fig. 6). The exogenous presence of Xyn11A in the leaves complements the lack of the protein in the mutant N23, since the differences between the wild-type and the mutant disappear. Again, this enhancing effect of Xyn11A is independent of the xylanase activity as the same effect could be seen with the Xyn11A protein devoid of xylan hydrolyzing ability.
A 30-amino acids peptide in the Xyn11A surface mediates necrotizing activity and binding to plant cell membrane
The necrotizing activity of the EIX xylanase from T. viride was previously mapped to the peptide TKLGE in the enzyme's surface [23]. However, this peptide is not present in Xyn11A, and is substituted by the peptide TEIGS (residues 139 to 143 in the immature protein) (Fig. 7A). The evidences presented by Rotblat et al. [23] to sustain the role of TKLGE were mainly two: first, affinity purified antibodies against the peptide blocked EIX necrotizing activity and its binding to plant cells, and second, mutant EIX in which TKLGE had been substituted by VKGT lost the necrotizing activity, but not the xylanase activity. From our point of view, it may also be possible therefore that the antibody binding, or the mutation of TKLGE, blocks the function of a bigger necrotizing epitope of which TKLGE is a part. In this respect, it is interesting that TEIGS in Xyn11A is followed by a region of 6 amino acids, VTSDGS, that is very well conserved in family 11 of glycosyl hydrolases and is located also on the enzyme surface (Fig. 7C and 7D). VTSDGS is perfectly conserved in the 3 xylanases that have been shown to induce necrosis (Fig. 7A), those of T. viride, T. reesei, and B. cinerea. The analysis of the alignment of 308 members of the Pfam family "Glycosyl hydrolases family 11", which are all putative xylanases, revealed that these 6 amino acids are also well conserved across the family. The first five are present in more than half of the proteins and the dipeptide Asp-Gly is present in virtually all members (Fig. 7B and 7C). The recognition by plants of a very well conserved epitope in family-11 xylanases would agree with the idea that pathogen associated molecular patterns recognized by the plant immune system should be, in principle, conserved microbial features [24]. We expressed in Escherichia coli a 30-aa region comprising two consecutive beta-sheets on the enzyme surface, one of which displays the region TEIGSVTSDGS (Fig. 7D). This peptide was expressed both as a fusion to the green fluorescent protein (GFP) (either at the N-terminus or at the C-terminus) and by itself, by using the pRSET series of expression vectors (Invitrogen, http://www.invitrogen.com). The three proteins were then purified with Nickel columns and infiltrated in tomato leaves to assay their elicitation ability (Fig. 7E). The two GFP fusion proteins were able to induce necrosis when infiltrated on leaves, while infiltration with GFP alone (Roche, http://www.roche-applied-science.com) dissolved in the same buffer, or with the buffer alone, did not show any effect. These results clearly indicate that the 30-aa epitope is sufficient to induce a response in the plant leading to the cell death. However, the epitope by itself did not cause any response (Fig. 7E). This difference in the activities of the peptide and its fusion with GFP may be attributed to a reduced stability of the isolated peptide or may indicate that the eliciting molecule needs to have a minimum size in order to produce any effect.
We used tobacco spheroplasts to check the binding of the necrotizing epitope-GFP fusion proteins to the cellular membrane. Spheroplasts from Nicotiana tabacum cv. Havana were mixed with the two fusion proteins (or GFP alone as a negative control), incubated for 30 min at room temperature, and finally examined by fluorescence microscopy (Fig. 8). We could observe for both epitope-GFP fusions the appearance of green fluorescence in the cells, which could not be observed for the spheroplasts treated with GFP alone or for the untreated ones, indicating that the 30-aa region is sufficient for binding to the plant surface.