ABSTRACT

Fungal pathogens usually have multiple genes that encode extracellular hydrolytic enzymes that may degrade the physical barriers in their hosts during the invasion process. Nectria hematococca, a plant pathogen, has two inducible pectate lyase (PL) genes (pel) encoding PL that can help degrade the carbohydrate barrier in the host. pelA is induced by pectin, whereas pelD is induced only in planta. We show that the disruption of either the pelA or pelD genes alone causes no detectable decrease in virulence. Disruption of both pelA and pelD drastically reduces virulence. Complementation of the double disruptant with pelD gene, or supplementation of the infection droplets of the double disruptant with either purified enzyme, PLA, or PLD, caused a recovery in virulence. These results show that PL is a virulence factor. Thus, we demonstrate that disruption of all functionally redundant genes is required to demonstrate the role of host barrier-degrading enzymes in pathogenesis and that dismissal of the role of such enzymes based on the effects of single-gene disruption may be premature.

Cutin monomers, generated by the low levels of constitutively expressed cutinase, induce high levels of cutinase that can help pathogenic fungi to penetrate into the host through the cuticle whose major structural polymer is cutin. We cloned three highly homologous cutinase genes, cut1, cut2, and cut3, from Fusarium solani f. pisi (Nectria haematococca). Amino acid sequence deduced from the nucleotide sequence of cut1 and cut2/3 matched with that of the peptides from cutinase 1 and cutinase 2, respectively, isolated from F. solani pisi grown on cutin as the sole carbon source. Induction of ␤-glucuronidase gene fused to the promoters of the cutinases integrated into F. solani pisi genome indicates that cut2 is constitutively expressed and induced under starvation, whereas cut1 is highly induced by cutin monomers. A palindrome binding protein (PBP) previously cloned binds only to palindrome 1 of cut1 promoter but not palindrome 1 of cut2/3 which contains two base substitutions. PBP is thought to interfere with the binding of CTF1␣, the transcription factor involved in induction, to cut1 promoter and thus keep cut1 gene repressed until induced by cutin monomers. Because PBP cannot bind palindrome 1 of cut2, this gene is not repressed. CTF1␣ does not transactivate cut2 promoter. A new Cys 6 Zn 2 motif-containing transcription factor, CTF1␤, that binds palindrome 2 was cloned and sequenced. In yeast, CTF1␤ transactivates cut2 promoter but not cut1 promoter unless its palindrome 1 is mutated, unlike CTF1␣ which transactivates cut1. Thus, CTF1␤ is involved in the constitutive expression of cut2 that causes production of low levels of cutin monomers that strongly induce cut1 using CTF1␣ as the transcription factor.Fungal infection of plants can be assisted by extracellular cutinases that help the pathogen penetrate through the outermost cuticular barrier of the host (1, 2). Conidia of highly virulent pathogens, which can directly penetrate through the cuticle, have low levels of cutinase that release small amounts of cutin monomers when the conidia contact the host surface (3). These monomers transcriptionally activate the expression of an inducible cutinase gene that is responsible for the production of high levels of cutinase that assist the infection peg to gain entry into the host through the cuticle (4). A cis element essential for the inducible expression of cutinase gene was found to be located at Ϫ159 bp in the promoter of this gene (5) in Fusarium solani f. pisi (Nectria haematococca). In this region, two overlapping palindromes were found. Palindrome 2 was found to be necessary for the inducible cutinase gene expression (5). A protein that binds the palindromic region, called palindrome binding protein (PBP), 1 (6) and a cutinase transcription factor 1␣ (CTF1␣), which selectively binds palindrome 2 and transactivates the cutinase promoter (7), have been cloned. CTF1␣, a 101-kDa protein, contains a Cys 6 Zn 2 binuclear cluster motif, sharing homology to the Cys 6 Zn 2 binuclear cluster DNA-binding domains of tr...

Hard-surface contact primes the conidia of Colletotrichum gloeosporioides to respond to plant surface waxes and a fruit-ripening hormone, ethylene, to germinate and form the appressoria required for infection of the host. Our efforts to elucidate the molecular events in the early phase of the hard-surface contact found that EGTA (5 mM) and U73122 (16 nM), an inhibitor of phospholipase C, inhibited (50%) germination and appressorium formation. Measurements of calmodulin (CaM) transcripts with a CaM cDNA we cloned from C. gloeosporioides showed that CaM was induced by hard-surface contact maximally at 2 h and then declined; ethephon enhanced this induction. The CaM antagonist, compound 48/80, completely inhibited conidial germination and appressorium formation at a concentration of 3 μM, implying that CaM is involved in this process. A putative CaM kinase (CaMK) cDNA of C. gloeosporioides was cloned with transcripts from hard-surface-treated conidia. A selective inhibitor of CaMK, KN93 (20 μM), inhibited (50%) germination and appressorium formation, blocked melanization, and caused the formation of abnormal appressoria. Scytalone, an intermediate in melanin synthesis, reversed the inhibition of melanization but did not restore appressorium formation. The phosphorylation of 18- and 43-kDa proteins induced by hard-surface contact and ethephon was inhibited by the treatment with KN93. These results strongly suggest that hard-surface contact induces Ca2+-calmodulin signaling that primes the conidia to respond to host signals by germination and differentiation into appressoria.

Hydroxy fatty acids from plant cutin were shown previously to induce the expression of the cutinase gene via a palindromic sequence located at ؊159 base pairs of the cutinase gene in Fusarium solani f. sp. pisi (Nectria hematococca mating type VI). Of the two overlapping palindromes in this sequence, palindrome 2 was found to be essential for the inducibility of cutinase by hydroxy fatty acids. Screening of a phage expression library with the concatenated palindrome 2 as probe detected a distinct cDNA clone encoding a polypeptide designated cutinase transcription factor 1␣ (CTF1␣) with a calculated molecular weight of 101,109. This protein contains a Cys 6 Zn 2 binuclear cluster motif sharing homology to the Cys 6 Zn 2 binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S. carlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nidulans, and A. flavus. CTF1␣, expressed in Escherichia coli, showed specific binding to the palindrome 2 DNA fragment but not to palindrome 1 or mutant palindrome 2 DNA fragments, suggesting specific binding of CTF1␣ to palindrome 2. When CTF1␣ was expressed as a fusion protein with the nuclear localization sequence of SV40 in yeast, it transactivated the native cutinase promoter fused to the chloramphenicol acetyl transferase (cat) gene. Mutation of palindrome 2 but not palindrome 1 abolished this transactivation. Thus, CTF1␣ positively acts in vivo by binding selectively to palindrome 2 of the cutinase gene promoter.

Differentiation of fungal conidia of phytopathogens into the infection structure, appressorium, requires contact with a hard surface and host signals. The molecular signaling involved in the induction of this differentiation is poorly understood. We report the cloning of a mitogen-activated protein kinase kinase (MEK), CgMEK, from Colletotrichum gloeosporioides and its role in the induction of these developmental processes involved in pathogenesis. Disruption of CgMEK1 resulted in the loss of its ability to form appressoria in response to the host's signals and a loss of virulence. Results of confocal microscopic examination of germinating conidia of the gene-disrupted mutants were similar to those for wild-type conidia treated with an MEK inhibitor, suggesting that CgMEK1 is involved in two developmental processes in the differentiation into appressorium: (1) polarized cell division, with the preferential increase in F-actin in one of the daughter nuclei after nuclear division and the formation of septum; and (2) differentiation of the germ tube into an appressorium. CgMEK1 is required for the differentiation. INTRODUCTIONMany phytopathogenic fungi are known to require hard surface contact of conidia before they can differentiate into infection structures called appressoria (Emmett and Parbery, 1975; Xiao et al., 1997). Physical or chemical signals from the host are also known to be required for this differentiation (Grover, 1971;Lapp and Skoropad, 1978; Parbery and Blakeman, 1978; Staples and Hoch, 1987;. Anthracnose disease, caused by the Colletotrichum (Gloeosporium) or the Glomerella group, is very common and destructive of numerous crop and ornamental plants worldwide. The conidia of Colletotrichum gloeosporioides , a causal agent of anthracnose disease on fruits, need a hard surface contact for 2 hr before they can differentiate into appressoria in response to host signals such as surface wax and the ripening hormone ethylene (Flaishman and Kolattukudy, 1994;Flaishman et al., 1995;. Protein phosphorylation is involved in this signal-induced process of appressorium formation because inhibition of protein phosphorylation by kinase inhibitors severely inhibits the appressorium formation induced by ethylene and host wax. The proteins phosphorylated in response to treatment with ethylene and wax include those similar in size to the mitogen-activated protein kinase, MAP kinase (MAPK;Flaishman et al., 1995).MAPK signaling pathways play a pivotal role in sensing extracellular signals and relaying the signals to control gene expression. (Nishida and Gotoh, 1993;Herskowitz, 1995). They involve three sequentially acting kinases-MAPK, MAPK kinase (MEK), and MEK kinase (MEKK) (Marshall, 1994)-the primary structures of which are remarkably conserved among eukaryotes from Saccharomyces cerevisiae to Homo sapiens . MAPK pathways are involved in a variety of developmental processes in yeast and filamentous fungi (Madhani and Fink, 1998a, 1998b). MAPKs have been cloned from phytopathogenic fungi (Li et al., 1997;Hamer an...

Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen, Colletotrichum gloeosporioides, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode Colletotrichum hard-surface induced proteins are designated chip genes. In this study, we report the characterization of CHIP2 and CHIP3 genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. The CHIP2 product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The CHIP3 product would be a nine-transmembrane-domain-containing protein. RNA blots showed that CHIP2 and CHIP3 are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressoriumforming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting that C. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.

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