The use of green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of 'Spadona' pear (Pyrus communis L.).

An efficient and reproducible system for Agrobacterium-mediated transformation of the pear (Pyrus communis L.) cultivar Spadona was developed. Leaf explants of in vitro propagated plants were cocultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504, carrying the uidA-intron and nptII genes. Under selective conditions, 5% of the plantlets regenerated and were positively stained for GUS. However, most of the GUS-positive plants re-callused and subsequently died, leaving only 0.3-0.8% of these plantlets to reach maturity. In order to identify transformed shoots at early stages of regeneration, we introduced the green fluorescent protein (GFP) into the pear cultivar Spadona using the plasmid PZP carrying the nuclear-targeted GFP and nptII genes. High expression levels of GFP were detected in transgenic cells as early as 7 days after transformation. GFP marked-callii and transformed plants were observed after 14 and 24 days, respectively. Fluorescence microscopy screening of transformed plant material, under the selection of kanamycin, increased the transformation frequency to 3.0-4.0%. We conclude that the introduction of GFP improves the selection of transformed plants of Spadona pear.

First Report of Pythium myriotylum on Lemna gibba in Israel.

Much interest has focused on duckweed as an alternative food source. Commercial production of Lemna gibba was initiated in Israel in 1991. During the summers of 1994 and 1995, Lemna gibba was grown commercially in covered ponds. In these two seasons, the plants were found to be infected by Pythium myriotylum, which has previously been reported as a pathogen of duckweed (1). In the ponds where the plants were held at high density, small, wilted, white patches of plants were recognized in early June. By the end of the month over 70% of the plants were infected and died. Microscopic examination of infected plants showed the presence of fungal appressoria and mycelial growth. Fungi were isolated from the plants onto potato dextrose agar (PDA). Ten isolates grown on PDA for 7 days were used to inoculate healthy plants. After 12 days, symptoms developed on plants at 28 or 32°C. Symptoms were similar to those observed on pond-grown plants. Plants maintained at 17 or 22°C did not become infected. The results from the pond and laboratory work showed that high plant density and temperature above 24°C were necessary for infection, and infection takes place by hyphal elements that spread in the water. This is the first report of Pythium myriotylum on Lemna gibba in Israel. Reference: (1) E. Rejmankova et al. Veröff. Geobot. Inst. Eidg. Tech. Hochsch. Stift. Rübel, Zür. 87:178, 1986.

Surface signaling in pathogenesis.

Surface signaling plays a major role in fungal infection. Topographical features of the plant surface and chemicals on the surface can trigger germination of fungal spores and differentiation of the germ tubes into appressoria. Ethylene, the fruit-ripening hormone, triggers germination of conidia, branching of hyphae, and multiple appressoria formation in Colletotrichum, thus allowing fungi to time their infection to coincide with ripening of the host. Genes uniquely expressed during appressoria formation induced by topography and surface chemicals have been isolated. Disruption of some of them has been shown to decrease virulence on the hosts. Penetration of the cuticle by the fungus is assisted by fungal cutinase secreted at the penetration structure of the fungus. Disruption of cutinase gene in Fusarium solani pisi drastically decreased its virulence. Small amounts of cutinase carried by spores of virulent pathogens, upon contact with plant surface, release small amounts of cutin monomers that trigger cutinase gene expression. The promoter elements involved in this process in F. solani pisi were identified, and transcription factors that bind these elements were cloned. One of them, cutinase transcription factor 1, expressed in Escherichia coli, is phosphorylated. Several protein kinases from F. solani pisi were cloned. The kinase involved in phosphorylation of specific transcription factors and the precise role of phosphorylation in regulating cutinase gene transcription remain to be elucidated.

Cloning of a gene expressed during appressorium formation by Colletotrichum gloeosporioides and a marked decrease in virulence by disruption of this gene.

Appressorium formation in germinating Colletotrichum gloeosporioides is induced by the surface wax of its host. One of the genes expressed uniquely in C. gloeosporioides during appressorium formation induced by the host signal has been designated cap20, and this gene and its cDNA were cloned and sequenced. Nucleotide sequences of both revealed an open reading frame that could encode a 183-amino acid polypeptide that did not have significant homology with any known proteins. Reverse transcriptase-polymerase chain reaction detected cap20 gene transcripts at the infection front on the surface and within tomato fruits infected by C. gloeosporioides. Gene-disrupted mutants incapable of expressing cap20 showed a drastically decreased virulence on avocado and tomato fruits. These results suggest that cap20 plays a significant role in the infection of the host.

Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea.

Fusarium solani f sp pisi (Nectria haematococca) isolate 77-2-3 with one cutinase gene produced 10 to 20% of the cutinase produced by isolate T-8 that has multiple cutinase genes, whereas cutinase gene-disrupted mutant 77-102 of isolate 77-2-3 did not produce cutinase. On the surface of pea stem segments, lesion formation was most frequent and most severe with T-8, less frequent and less severe with 77-2-3, and much less frequent and much milder with the gene-disrupted mutant. Microscopic examination of the lesions caused by the mutant strongly suggest that it penetrated the host mostly via the stomata. In seedling assays, 77-2-3 caused severe lesions on every seedling and stunted growth, whereas the mutant showed very mild lesions on one-third of the seedlings with no stunting. Thus, cutinase gene disruption resulted in a significant decrease in the pathogenicity of F. s. pisi on pea.

Timing of fungal invasion using host's ripening hormone as a signal.

In many postharvest fruit diseases, fungi remain latent until the fruit ripens. How the fungus times its infection at ripening of the host is not known. We have found that the volatiles produced by the climacteric tomato, avocado, and banana fruits induce germination and appressorium formation in Colletotrichum gloeosporioides and Colletotrichum musae. Exposure of the spores of these fungi to ethylene, the host's ripening hormone, at