Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection

The presence of the fungal pathogen, Botrytis cinerea, in the vineyard causes reductions in both quality and quantity of grapes and wine. Because proteins are involved in the foam stabilization of sparkling wines, we have undertaken, for the first time, a thorough proteomic analysis of two champagne base wines prepared with either healthy or botrytized Chardonnay grapes, using two-dimensional electrophoresis (2DE) coupled with immunodetection and tandem mass spectrometry. Most of the identified proteins were from grape origin: invertase and pathogenesis-related (PR) proteins. The disappearance of numerous grape proteins was observed in the botrytized wine, suggesting that they were probably degraded or even repressed or the result of a differential expression of grape proteins upon fungal infection. On the other hand, two pectinolytic enzymes secreted by B. cinerea were found in the botrytized wine.     

Transgenic expression of plant chitinases to enhance disease resistance

Crop plants have evolved an array of mechanisms to counter biotic and abiotic stresses. Many pathogenesis-related proteins are expressed by plants during the attack of pathogens. Advances in recombinant DNA technology and understanding of plant–microbe interactions at the molecular level have paved the way for isolation and characterization of genes encoding such proteins, including chitinases. Chitinases are included in families 18 and 19 of glycosyl hydrolases (according to www.cazy.org) and they are further categorized into seven major classes based on their aminoacid sequence homology, three-dimensional structures, and hydrolytic mechanisms of catalytic reactions. Although chitin is not a component of plant cell walls, plant chitinases are involved in development and non-specific stress responses. Also, chitinase genes sourced from plants have been successfully over-expressed in crop plants to combat fungal pathogens. Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.     

Coordinate Accumulation of Antifungal Proteins and Hexoses Constitutes a Developmentally Controlled Defense Response during Fruit Ripening in Grape

During ripening of grape (Vitis labruscana L. cv Concord) berries, abundance of several proteins increased, coordinately with hexoses, to the extent that these became the predominant proteins in the ovary. These proteins have been identified by N-terminal amino acid-sequence analysis and/or function to be a thaumatin-like protein (grape osmotin), a lipid-transfer protein, and a basic and an acidic chitinase. The basic chitinase and grape osmotin exhibited activities against the principal grape fungal pathogens Guignardia bidwellii and Botrytis cinerea based on in vitro growth assays. The growth-inhibiting activity of the antifungal proteins was substantial at levels comparable to those that accumulate in the ripening fruit, and these activities were enhanced by as much as 70% in the presence of 1 m glucose, a physiological hexose concentration in berries. The simultaneous accumulation of the antifungal proteins and sugars during berry ripening was correlated with the characteristic development of pathogen resistance that occurs in fruits during ripening. Taken together, accumulation of these proteins, in combination with sugars, appears to constitute a novel, developmentally regulated defense mechanism against phytopathogens in the maturing fruit.Plants have evolved a number of strategies to resist fungal infection. One strategy involves the accumulation of defense proteins that have direct inhibitory activity against the hyphae and/or germinating spores of the pathogen. Among these are PR proteins including chitinases (PR-3 family), thaumatin-like proteins (PR-5 family), and nsLTPs. Typically, these antifungal proteins are expressed constitutively at low levels in cells and accumulate in response to fungal attack or in response to other inducers of acquired resistance (Uknes et al., 1992). Reproductive organs are apparently an exception to induced acquired resistance. Presumably, the importance of flowers and ovaries to the maintenance of the species has mandated that reproductive organs acquire pathogen resistance during development. Developmentally regulated expression of PR proteins has been observed in floral organs (Lotan et al., 1989; Neal et al., 1990), including numerous examples of defensive gene mRNA accumulation in fruits. However, comparatively few data are available that interrelate developmental accumulation of antifungal proteins and the acquisition of resistance against fruit pathogens (Fils-Lycaon et al., 1996; Meyer et al., 1996).Another physiological adaptation of plants that affects fungal pathogenesis, but one that has received considerably less attention, is the accumulation of sugars. Results from studies of several host/pathogen systems have implicated accumulation or depletion of sugars in resistance to fungal infection (VanderPlank, 1984). Increased susceptibility to fungi in sugar-depleted vegetative tissues, a phenomenon termed “sink-induced loss of resistance,” has been documented in tomato (Horsfall, 1975), cotton (Eaton and Rigler, 1946), and maize (Holbert et al., 1935). Conversely, moderate levels of sugars can enhance colonization rates of some fungal pathogens, presumably because these are important sources of C for the microbes (Mains, 1917). However, higher levels of sugars can reverse this effect, leading to decreased susceptibility, a phenomenon termed “high-sugar resistance” (Horsfall and Dimond, 1957). This reversal of susceptibility has been suggested to occur as a result of high sugar levels furnishing an osmotic challenge to the fungi and suppressing their colonization of the plant (VanderPlank, 1984).We report here that accumulation of hexoses in grape (Vitis labruscana L. cv Concord) berries is accompanied by a developmental-stage-specific increase in a suite of proteins that are homologous to proteins known to be antifungal determinants. These proteins have been identified as a thaumatin-like protein (Salzman et al., 1994) (here we have named it GO), chitinases (both CBC and AC forms), and a nsLTP. Physiological levels of GO or CBC assayed in vitro alone or in combination with physiological levels of Glc exhibited individual and/or combinatorial activities against the important grape pathogens Guignardia bidwellii, the causal agent of black rot, and Botrytis cinerea. The interaction of the antifungal proteins and hexoses appears to constitute a developmentally regulated defense mechanism to restrict fungal pathogen infection as seeds are maturing in ripening berries.     

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20–30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.     

The diversity of pathogenesis-related proteins decreases during grape maturation

It was recently shown that wines contain typically a huge diversity of structurally similar polypeptides that exhibit a high degree of homology to pathogenesis-related (PR) proteins. This observation suggested the existence of one or a few precursors in mature grapes, common to most or all the wine PR proteins. Limited proteolysis and chemical modification of the precursor(s) during fruit ripening and winemaking could then generate the large number of distinct wine polypeptides. However, the patterns of PR proteins extracted from grape berries regularly harvested from the onset of development until maturity did not confirm the previous hypothesis. Two different methodologies, involving 2-D immunoblotting and a combination of FPLC cation/anion exchange chromatographies with 1-D immunoblotting, indicate that the total concentration of PR proteins is increased but its diversity is reduced from the early stages of berry development until maturity. These results indicate that PR proteins are synthesized in a wide variety of forms from the early stages of grape development, eliminating the hypothesis previously formulated on the existence of one or few precursors common to the wine proteins.     

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20-30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.     

Identification and functionality prediction of pathogenesis‐related protein 1 from legume family

The production and accumulation of pathogenesis‐related (PR) proteins in plants is one of the important responses to biotic and abiotic stress. Large number of identified PR proteins has been categorized into 17 functional families based on their structure, phylogenetics, and biological activities. However, they are not widely studied in legume crops. Using 29 PR1 proteins from Arabidopsis thaliana, as query, here we have predicted 92 candidate PR1 proteins through the PSI‐BLAST and HMMER programs. These candidate proteins were comprehensively analyzed with, multiple sequence alignment, domain architecture studies, signal peptide, and motif extraction followed by phylogenetic analysis. Further, response of two candidate PR1 proteins from chickpea against Fusarium oxysporum f.sp.ciceri attack was validated using qRT‐PCR followed by their 3D structure prediction. To decipher mode of action for PR1s, docking of pathogen extracellular matrix components along with fungal elicitors was performed with two chickpea PR1 proteins. Based on these findings, we propose carbohydrate to be the unique pathogen‐recognition feature for PR1 proteins and β‐glucanase activity via β‐glucan binding or modification.     

Grape and apple wines volatile fermentation products and possible relation to spoilage

The main volatile by-products of the alcoholic fermentation of grape wine, cider and apple pulp wine were investigated to determine if any correlated with spoilage resistance in the latter two. Spoilage was visually detected after seven days in low-alcohol grape wine in comparison to 11 and 16 days in cider and apple pulp wine, respectively. Acetaldehyde, ethyl acetate, methanol, propanol, isobutanol and amyl alcohols were the main fermentation by-products detected in all three wines. There were highest concentrations of acetaldehyde, ethyl acetate, methanol and propanol in grape wine and, therefore, these by-products could not be implicated in spoilage resistance in apple wines. Increased concentrations of isobutanol and amyl alcohols, however, in cider and apple pulp wine in comparison to grape wine might have been the reason for spoilage resistance in the apple wines.     

Proteomic Analysis of Sauvignon Blanc Grape Skin,Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR proteins in berries at harvest.     

The role of plant defence proteins in fungal pathogenesis

It is becoming increasingly evident that a plant–pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillion-year evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant–pathogen interaction is an essential goal for disease control in the future.     

Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.     

Synthesis of Stress Proteins in Tobacco Leaves

Pathogenesis-related proteins (PR proteins), which are knownto be induced in tobacco leaves in response to infection withtobacco mosaic virus (TMV), were isolated by a simple procedureinvolving ammonium sulfate fractionation and preparative gelelectrophoresis. A rabbit antibody to one of the purified PRproteins, designated as PR1a, also reacted with two other PRproteins, designated as PR1b and PR1c in double immunodiffusiontests. Quantitative analysis of these proteins using rocketimmunoelectrophoresis with the antibody showed that they wereinduced not only by infection with TMV but also by mechanicalinjury and osmotic stress at 20?C, although not at 30?C. Basedon these findings, we propose that these proteins be called"stress proteins" rather than "pathogenesis-related proteins." (Received October 23, 1984; Accepted January 18, 1985)     

Nontarget and ecological effects of transgenically altered disease resistance in crops -possible effects on the mycorrhizal symbiosis

The ability to increase crop disease resistance by using transgenic (TG) means has recently been demonstrated for several crops. The current TG procedures alter the temporal expression of transgene pathogenesis-related (PR) proteins, so that the usually inducible PR proteins are expressed constitutively in the foreign host. The constitutive expression of the transgene PR protein chitinase is believed to increase the host's nonspecific basic resistance to pathogens. A potential nontarget effect of constitutively expressing chitinase may be a decrease in the activity of beneficial microbes, especially vesicular-arbuscular mycorrhizal fungi. The decrease in activity of mycorrhizal fungi is related to reduced susceptibility of TG plant roots to colonization by these fungi, which is in turn associated with lysis of fungal cell walls by the constitutively expressed chitinase. An argument is presented that use of TG means to alter the temporal expression of PR proteins ignores a legacy of past evolutionary trade-offs in vascular plants. A major nontarget effect of expressing transgene chitinase is a reduction in the susceptibility of roots to colonization by mycorrhizal fungi. This reduction in mycorrhizal susceptibility occurs without alteration of the mycorrhizal dependence of the host on symbiont-supplied nutrients. Data are presented in support of this contention that demonstrate a strong negative association between host pathogen resistance and mycorrhizal colonization. An ecological consequence of reducing mycorrhizal colonization is a decrease in the soil's mycorrhizal propagule reserve that diminishes the next crop's production, especially under low-input cropping practices. A further consequence that has both ecological and evolutionary outcomes is the escape of the transgene for improved pathogen resistance into wild populations. By increasing a crop's disease resistance by TG means, we may inadvertently be creating a ‘super weed’ when the TG plant or the transgene escapes into wild relatives through hybridization. Hybridization of wild relatives with TG plants would be especially relevant for crops, such as sugar beet, rapeseed, and many modern cereal cultivars that have close relatives in the wild but have a relatively low requirement for symbiont supplied nutrients or are nondependent.     

Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants

Pathogen attack represents a major problem for viticulture and for agriculture in general. At present, the use of phytochemicals is more and more restrictive, and therefore it is becoming essential to control disease by having a thorough knowledge of resistance mechanisms. The present work focused on the trans-regulatory proteins potentially involved in the control of the plant defence response, the WRKY proteins. A full-length cDNA, designated VvWRKY1, was isolated from a grape berry library (Vitis vinifera L. cv. Cabernet Sauvignon). It encodes a polypeptide of 151 amino acids whose structure is characteristic of group IIc WRKY proteins. VvWRKY1 gene expression in grape is regulated in a developmental manner in berries and leaves and by various signal molecules involved in defence such as salicylic acid, ethylene, and hydrogen peroxide. Biochemical analysis indicates that VvWRKY1 specifically interacts with the W-box in various nucleotidic contexts. Functional analysis of VvWRKY1 was performed by overexpression in tobacco, and transgenic plants exhibited reduced susceptibility to various fungi but not to viruses. These results are consistent with a possible role for VvWRKY1 in grapevine defence against fungal pathogens.     

Induction of β-1,3-glucanase and chitinase in Vigna aconitifolia inoculated with Macrophomina phaseolina

Pathogenesis-related (PR) proteins are induced in response to pathogen attack. In the present study, the induction of PR proteins in response to the fungal pathogen Macrophomina phaseolina was investigated in 15-day- and 1-month-old plants of Vigna aconitifolia with resistant and susceptible cultivars. Inoculation of the fungal pathogen resulted in the enzyme activity gradually increased throughout the experimental period of 168 h compared to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in the resistant FMM-96 cultivar as compared to susceptible RM0-40 and CZM-3 cultivars. Furthermore, the western blot analysis revealed the presence of 33- and 30-kDa bands of β-1,3-glucanase and chitinase in induced moth bean plants, respectively. The possible implications of these findings as part of the general defense response of moth bean plants against the fungal pathogen (M. phaseolina) have been discussed.     

Qualität und Resistenz

Summary The quality of wines and unfermented grape juices depends, among others, on their biotic value, checked by biological tests. This value shows a positive correlation with the degree of resistance of the plants to botanical and zoological parasites and the pathogenic effects of their products. Wines and juices of low biotic value fromVitis varieties and species with high resistance were fed to hens and chicks. Treated chicks from untreated parents and untreated chicks from treated parents developed certain anomalies, partly expressed by malformation of the legs and by cramplike conditions attributable to deficient nervous function. The relation between the changes caused in the yolk and egg white by specific biostatica in wines and grape juices fed to fowl and the indicators of grape resistance are discussed.     

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by <Emphasis Type="Italic">Alternaria alternata</Emphasis> apple pathotype

Alternaria blotch, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of serious pathogen of apples. In order to better understand the molecular mechanisms that underlie the defense responses of apple resistance to Alternaria blotch disease, a comparative proteomic approach was applied to analyze of susceptible and resistant apple cultivars response to A. alternata AP infection using iTRAQ (isobaric tags for relative and absolute quantitation) technique. A total of 4225 proteins were identified, and 1226 proteins were quantified. Of the quantified proteins, 280 and 34 expressed differentially (fold change >1.5) at 72 h post-infection (HPI) in the susceptible (“Starking Delicious”) and the resistant (“Jonathan”) apple cultivars, respectively, compared with mock-inoculated controls. Most of the differentially expressed proteins (DEPs) were associated with host plant resistance to pathogens, including signal transduction, stress and defense, and photosynthesis metabolism. Among these proteins, beta-1,3-glucanase(PR2), thaumatin-like protein (PR5), and lipoxygenase were found in both susceptible and resistant hosts. However, endochitinase and (+)-neomenthol dehydrogenase were only detected in the resistant cultivar and increased in abundance in response to the pathogen attack. To study the role of pathogenesis-related (PR) proteins in the early infection process, their expressions at 6, 18, 36, and 72 HPI were analyzed by western blot. It showed that PR5 were accumulated to a high level at 6 HPI in “Jonathan,” while cannot be detected in “Starking Delicious” until 18 HPI. The above results suggested that endochitinase and (+)-neomenthol dehydrogenase, as well as PR5 which exerts function at early stage, play important roles in apple plant against A. alternata AP infestation.     

Endophytic Fungal strains Specifically Modified the Biochemical Status of Grape Cells

Previously, specific interactions in morphology were observed between grape cells and endophytic fungal strains during a dual culture experiment. However, the biochemical impacts of these fungal endophytes on grape cells is also expected due to their potential application in grape quality management. After assessed multiple physiochemical traits to those grape cells which have co-cultured with different endophytic fungal strains in this study, and found the presence of fungal endophytes obviously triggered ROS stress responses in grape cells, and the biochemical status in grape cells were differentially modified by different fungal strains. In those tested endophytic fungal strains, RH37 (Epicoccum sp.), RH6 (Alternaria sp.), RH32 (Alternaria sp.) and RH34 (Trichothecium sp.) conferred greater metabolic impacts on grape cells. And soluble protein (SPr), total flavonoids (TF), total phenols (TPh) and malondialdehyde (MDA) on the other hand, were sensitive biochemical parameters which can be influenced in greater ranges than other detected parameters. Most interestedly, fungal endophytes shaped metabolites patterns in grape cells during the dual culture appeared fungal genus/species/strain-specificity. The work confirmed the significance of fungal endophytes in grape metabolic regulation and elucidated the possibility to purposely manage grape quality using tool of fungal endophytes.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.