This study constitutes the first genome-wide effort to understand the molecular basis of a host-X. fastidiosa interaction in Vitis. Twelve forward and reverse suppression subtractive cDNA libraries from two genotypes (resistant and susceptible) for three different tissues and 10 different stages of Pierce's disease development were constructed to identify spatial and temporal transcriptional changes resulting from X. fastidiosa infection. Because a whole Vitis genome sequence is not yet been completed, ESTs could serve as an efficient alternative approach to the discovery of novel genomic information. Out of the 1,942 non-redundant ESTs that were cloned in this study, about 33% were found to be unique, demonstrating the effectiveness of the experimental design and the construction strategy utilized for these SSH libraries. RT-PCR analysis of seven out of the eight selected ESTs from SSH confirmed their differential expression under the test conditions. Five out of the six transcripts showed up regulation in the tissue types and condition from which they were cloned. However, the number of transcripts that were cloned for each of these ESTs (based on the ESTs that were used in generating the contig) was several folds lower than their original numbers (as indicated by the RT-PCR change values) in the RNA pool, indicating that suppression of the EST numbers that appeared in the final pool was effective. Furthermore, more than half (54%) of these sequences did not match the sequences in the GenBank and 508 were not reported in the Vitis EST database collection and are therefore unique contributions to the Vitis EST pool. A significant difference in the number and diversity of transcripts was observed in response to X. fastidiosa infection in the resistant vs. susceptible genotypes, suggesting host responses to infection are genotype dependent. The present study identified a group of transcripts that are regulated in response to X. fastidiosa infection and may represent the key elements in development of the defense response.
There was a significant reduction in transcript diversity, particularly in leaf tissues, in both the resistant and susceptible genotypes, after infection with X. fastidiosa
(Table ). This transcript variation was supported by the co-expression pattern of the ESTs with only 28% of the ESTs overlapping among the four classes and the rest being unique to each of those classes (Figure ). The large percentage of transcripts involved in ligand binding, carrier signal transduction, and defense response among the annotated transcripts from the inoculated tissue also supports the presumption that many of these transcripts are specifically involved in the X. fastidiosa
resistance response. These observations are consistent with previously reported studies on host-pathogen interactions [21
Among the three tissue types, comparisons between libraries from resistant and susceptible infected stem tissues produced the most interesting EST expression patterns. The resistant library had ESTs with primary cell wall modifying and metabolic enzymes and for known PR proteins such as β 1–3 glucanase.
Plant cell elongation depends on physical properties of the primary cell wall. The class of enzymes, called alternatively endo-xyloglucan transglycosylase (EXT) or xyloglucan endotransglycosylase (XET), modifies xyloglucan (XG) by cleavage and rejoining of the β(1–4)-XG backbone. Such activity can potentially alter cell size by loosening or tightening of the cell wall. Enzymes with XET activity have been identified in rapidly growing tissues from various plant species [23
] and multigene families related to XET have been identified [24
]. Expression of primary cell wall modifying ESTs in the RI stem library, suggest active modification and expansion of cell wall tissues. Such cell wall modifications have been hypothesized to be physical barriers to limit further pathogen invasion [26
]. Furthermore, expression of ESTs involved in cell metabolic activities might also reflect the pathogen's minor effect on tissue metabolism in these cells. The microarray comparative analysis study conducted by Bray [27
] indicated that the xyloglucan endotransglucosylase/hydrolases (XTHs) family of genes was down regulated under water deficit conditions in three independent experiments, supporting the non-water stressed nature of the RI plants.
Enhanced transcription of β 1–3 glucanase activity in grape has been previously associated with exogenous application of ethephon, an ethylene precursor [28
]. In a more recent study, Kortekamp [29
] found that PR-2 (β 1–3 glucanase) expression was associated with responses to Pseudoperenospora cubensis
infection in the resistant grape rootstock 'Gloire de Montpellier' (V. riparia
) compared to the susceptible cultivar 'Riesling' (V. vinifera
). EST expression in the susceptible stem library involved expression of a different class of PR proteins (PR-23S NP24 protein precursor and osmotin-like protein TPM-1) and also had different levels of seed storage and proteolytic EST expression, compared to their control tissues. Seed storage proteins such as legumins and vicillins are synthesized and accumulated during seed maturation and due to their regulation by agents such as abscisic acid, are associated with developing desiccation tolerance that occurs during seed maturation [30
]. Small protein ubiquitin (Ub) and the 26S proteosome, a 2-MDa protease complex, are key components of the proteolytic pathway [31
]. In response to pathogen attack, the Ub/26S proteosome pathway initiates programmed cell death to localize pathogen spread [31
]. Activation of proteolysis pathway ESTs in response to the pathogen attack has been documented previously [32
Some of the PR proteins such as chitinases and 14 kDa proline-rich protein ESTs were cloned only from resistant stem libraries. While ESTs, such as PR-10, were cloned from infected and control stem libraries of both susceptible and resistant selections. Previous reports in grape on PR-10 (intracellular proteins with unknown enzymatic function) expression point to its constitutive pre-infection role in pathogen defense [29
]. The previously described proline rich proteins or P-rich proteins in Arabidopsis
] and in Drosophila
] are known antimicrobial compounds. Further functional studies will be required to understand the specific role of these cloned PR proteins in resistant stem tissues during X. fastidiosa
Krivanek and Walker [2
] found that resistant stems host 60-fold fewer X. fastidiosa
cells than susceptible stems. The EST profiles produced here found unhindered metabolic activity in the resistant stem tissues and the occurrence of seed storage and proteolytic pathway proteins in the susceptible stem tissues, both suggesting the existence of a response to infection. Although PR protein expression was observed in the susceptible tissues, the nature of this expression was different since few of the PR proteins expressed in the susceptible tissues overlapped with those from resistant tissues. This finding suggests that even susceptible genotypes have a systemic and broad host defense response mechanism that responds to X. fastidiosa
infection, it does not prevent PD and must be augmented to achieve the resistance observed in 9621-67.
Four-way comparative analysis of the V. arizonica hybrid sequences with three other Vitis species contained in the GenBank EST collections (V. vinifera, V. shuttleworthii and V. aestivalis) revealed that 26% (508 ESTs) of the V. arizonica sequences were unique. There are 415 ESTs in common with V. vinifera (Unigene Built dated 04/13/06), 57 ESTs that were present in this set and the V. shuttleworthii set; and 24 ESTs that were also present in V. aestivalis set, but absent in the other two sets. In addition, there were 338 ESTs in common with the V. vinifera and V. shuttleworthii sets; 99 ESTs that were also present in V. vinifera and V. aestivalis sets, and 14 that were present also in V. shuttleworthii and V. aestivalis sets. The rest of the ESTs were found in all four sets.
This is the first study to display the extent of EST transcript diversity in grape after infection by X. fastidiosa
. A four-way comparative analysis found that each of the EST collections had an independent niche with varying degrees of overlap with the set produced from V. arizonica
. This study has identified likely molecular targets for developing PD resistant varieties and for characterizing their resistance genes. Based on the diversity and specificity of the presented EST cloning results, it is clear that stem tissue plays a prominent role in the X. fastidiosa
grape interaction, supporting observations by Krivanek and Walker [2
]. The generated ESTs with its unique collection will serve as an important addition to the grape transcript pool for further large scale expression studies.