The main molecular factors involved in the complex interactions occurring between plants (bean), two different fungal pathogens (Botrytis cinerea, Rhizoctonia solani) and an antagonistic strain of the genus Trichoderma were investigated. Two-dimensional (2-D) electrophoresis was used to analyze separately collected proteomes from each single, two- or three-partner interaction (i.e., plant, pathogenic and antagonistic fungus alone and in all possible combinations). Differential proteins were subjected to mass spectrometry and in silico analysis to search for homologies with known proteins. In the plant proteome, specific pathogenesis-related proteins and other disease-related factors (i.e., potential resistance genes) seem to be associated with the interaction with either one of the two pathogens and/or T. atroviride. This finding is in agreement with the demonstrated ability of Trichoderma spp. to induce systemic resistance against various microbial pathogens. On the other side, many differential proteins obtained from the T. atroviride interaction proteome showed interesting homologies with a fungal hydrophobin, ABC transporters, etc. Virulence factors, like cyclophilins, were up-regulated in the pathogen proteome during the interaction with the plant alone or with the antagonist too. We isolated and confidently identified a large number of protein factors associated to the multi-player interactions examined.

Tomato high pigment (hp) mutants are characterized by their exaggerated photoresponsiveness. Light-grown hp mutants display elevated levels of anthocyanins, are shorter and darker than wild-type plants, and have dark green immature fruits due to the overproduction of chlorophyll pigments. It has been proposed that HP genes encode negative regulators of phytochrome signal transduction. We have cloned the HP-2 gene and found that it encodes the tomato homolog of the nuclear protein DEETIOLATED1 (DET1) from Arabidopsis. Mutations in DET1 are known to result in con-stitutive deetiolation in darkness. In contrast to det1 mutants, tomato hp-2 mutants do not display any visible phenotypes in the dark but only very weak phenotypes, such as partial chloroplast development. Furthermore, whereas det1 mutations are epistatic to mutations in phytochrome genes, analysis of similar double mutants in tomato showed that manifestation of the phenotype of the hp-2 mutant is strictly dependent upon the presence of active phytochrome. Because only one DET1 gene is likely to be present in each of the two species, our data suggest that the phytochrome sig-naling pathways in which the corresponding proteins function are regulated differently in Arabidopsis and tomato.

Tomato high pigment ( hp ) mutants are characterized by their exaggerated photoresponsiveness. Light-grown hp mutants display elevated levels of anthocyanins, are shorter and darker than wild-type plants, and have dark green immature fruits due to the overproduction of chlorophyll pigments. It has been proposed that HP genes encode negative regulators of phytochrome signal transduction. We have cloned the HP-2 gene and found that it encodes the tomato homolog of the nuclear protein DEETIOLATED1 (DET1) from Arabidopsis. Mutations in DET1 are known to result in constitutive deetiolation in darkness. In contrast to det1 mutants, tomato hp-2 mutants do not display any visible phenotypes in the dark but only very weak phenotypes, such as partial chloroplast development. Furthermore, whereas det1 mutations are epistatic to mutations in phytochrome genes, analysis of similar double mutants in tomato showed that manifestation of the phenotype of the hp-2 mutant is strictly dependent upon the presence of active phytochrome. Because only one DET1 gene is likely to be present in each of the two species, our data suggest that the phytochrome signaling pathways in which the corresponding proteins function are regulated differently in Arabidopsis and tomato. INTRODUCTIONLight is a critical environmental signal controlling many aspects of plant development. For example, dark-grown plants display a typical etiolated morphology with elongated hypocotyls, closed apical hooks, and unexpanded cotyledons, whereas plants grown in the light have short hypocotyls, opened apical hooks, and expanded photosynthetically active cotyledons. Light is perceived by a series of photoreceptors that can detect light within a wide spectral range. The phytochromes are the best characterized of these photoreceptors and are able to intercept light primarily within the red and far-red regions of the spectrum (Furuya and Schäfer, 1996). They exist as multigene families, for example, PHYA to PHYE in Arabidopsis, and each phytochrome is likely to have a specific photoperceptory function during plant development (Quail et al., 1995). In addition, plants contain blue/UV-A-absorbing cryptochromes and UV-Babsorbing photoreceptors.Several models for light signal transduction in plants have been proposed. One has been deduced largely by microinjection experiments with tomato and involves G proteins, calcium, and cGMP (Bowler et al., 1994b;Mustilli and Bowler, 1997). Others are based on the genetic analysis of Arabidopsis mutants, such as deetiolated ( det ) and constitutively photomorphogenic ( cop ), which display characteristics of light-grown plants when grown in complete darkness, for example, reduced hypocotyl length, cotyledon opening and expansion, chloroplast development, and expression of light-induced genes (Chory et al., 1989(Chory et al., , 1996Deng et al., 1991;Chamovitz and Deng, 1996).Although several COP and DET genes have been identified, it is not clear how the activities of their gene products are regulated by phytochrome or by the calcium-an...

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.

Plant genetic engineering has long been considered a valuable tool to fight fungal pathogens because it would limit the economically costly and environmentally undesirable chemical methods of disease control. Ribosome-inactivating proteins (RIPs) are potentially useful for plant defense considering their antiviral and antimicrobial activities but their use is limited by their cytotoxic activity. A new gene coding for an RIP isolated from leaves of Phytolacca heterotepala was expressed in tobacco under the control of the wound-inducible promoter of the bean polygalacturonase-inhibiting protein I gene to increase resistance against different fungal pathogens, because an individual RIP isolated from P. heterotepala showed direct antifungal toxicity. Phenotypically normal transgenic lines infected with Alternaria alternata and Botrytis cinerea showed a significant reduction of leaf damage while reverse transcription-polymerase chain reaction and western analysis indicated the expression of the RIP transgene upon wounding and pathogen attack. This work demonstrates that use of a wound-inducible promoter is useful to limit the accumulation of antimicrobial phytotoxic proteins only in infected areas and that the controlled expression of the PhRIP I gene can be very effective to control fungal pathogens with different phytopathogenic actions.

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