We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves. P lant pathogenic fungi are a major threat for crop production worldwide. Several of these fungal pathogens depend on the formation of a specialized cell, called an appressorium, for the successful penetration of host plant surfaces (1, 2). This penetration process is typical of fungal leaf spots such as rice blast caused by the filamentous ascomycete Magnaporthe grisea (3). This disease is disseminated by water-splashed conidia (ref. 4; Fig. 1A). After their adhesion to the host leaf by means of a strong glue (ref. 5; Fig. 1B), conidia germinate and differentiate appressoria at the tips of their germ tubes (Fig. 1C). This developmental process is induced by chemical and physical stimuli from the host surface such as hardness, hydrophobicity, or cutin monomers (2, 3). This dome-shaped, thick-walled, and melanized cell (Fig. 1D) promotes the differentiation of a specialized hypha called the penetration peg that is involved in penetration of plant surfaces (ref. 6; pp in Fig. 1E). The differentiation of appressoria and penetration pegs is the result of complex morphogenetic processes that involve major developmental and metabolic changes (2,3,6). Inside the epidermal plant cell, the penetration peg differentiates into a bulbous infection hypha that invades the whole cell and colonizes the leaf.To isolate genes involved in this infection process, we generated a collection of M. grisea pathogenicity mutants by using plasmid-mediated insertional mutagenesis. This strategy was successful for the cloning of pathogenicity genes in Colletotrichum lindemuthianum (7), Ustilago maydis,** and M. grisea (8,9). In this study, we isolated punchless, a nonpathogenic mutant defective in penetration of plant surfaces. We show that the PLS1 gene is inactivated in punchless and encodes a putative membrane protein related to the tetraspanin family (10...

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Two major types of Frankia strains are usually recognized, based on the ability to sporulate in-planta: spore-positive (Sp+) and spore-negative (Sp-). We carried out a study of Sp+ and Sp- Frankia strains based on nodules collected on Alnus glutinosa, Alnus incana and Alnus viridis. The nodules were phenotyped using improved histology methods, and endophytic Frankia strain genotype was determined using a multilocus sequence analysis approach. An additional sampling was done to assess the relation between Sp+ phenotype frequency and genetic diversity of Frankia strains at the alder stand scale. Our results revealed that (i) Sp+ and Sp- Alnus-infective Frankia strains are genetically different even when sampled from the same alder stand and the same host-plant species; (ii) there are at least two distinct phylogenetic lineages of Sp+ Frankia that cluster according to the host-plant species and without regard of geographic distance and (iii) genetic diversity of Sp+ strains is very low at the alder stand scale compared with Sp- strains. Difference in evolutionary history and genetic diversity between Sp+ and Sp- Frankia allows us to discuss the possible ecological role of in-planta sporulation.