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.