Regulation of Gene Expression in Fungi for Breaching the Plant Host Barriers

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The plant cuticle, being the outermost barrier, is the first point of contact in the interaction between plants and fungal pathogens. The cuticle is composed of an insoluble biopolyester, cutin, and a complex mixture of soluble, non-polar lipids collectively called waxes. Cutin is the first structural barrier that the fungal infection peg must penetrate in order to infect the plant. Prior support from NSF was primarily responsible for the basic information obtained about the chemistry and biosynthesis of cutin, as well as the role of the cuticle in plant-fungus interaction. Cutinase secreted by the invading fungus helps it to attach to the host and penetrate the outer barrier. According to previously obtained evidence, when fungal conidia contact a plant surface, the small amount of constitutively expressed cutinase present on the conidia releases hydroxy fatty acid monomers and oligomers from cutin, and these soluble products cause induction of high levels of cutinase that helps the infection process. With cloned cutinase genes and transcription factors, it has been shown that in Nectria haematococca one transcription factor, CTF1b, regulates a constitutively expressed cutinase gene (cut2), and another transcription factor, CTF1a, is involved in cutin monomer induction of another cutinase gene, cut1. The mechanism by which hydroxy fatty acids or their oligomers induce cut1 remains to be elucidated. Cut1 induction involves a palindromic promoter element that contains 2 overlapping palindromes. Palindrome 2 is required for cut1 induction by cutin monomers. Palindrome binding protein (PBP) binds palindrome1 of cut1 and acts as a repressor that prevents CTF1a binding to palindrome 2. Since palindrome 1 of cut2 has 2 nucleotide substitutions, PBP cannot bind to it and suppress cut2 expression, and thus this gene is constitutively expressed. It is postulated that ctf1a induction by cutin monomers allows CTF1a to outcompete with PBP to bind cut1 promoter and cause cut1 induction. Cutin monomer or oligomer-stimulated phosphorylation of this cutin response element binding protein (CREBP) allows it to bind CRE and transcriptionally activate ctf1a gene. It is also possible that phosphorylation of PBP might weaken its binding to palindrome1 of cut1 and thus help relieve the suppression. This project will test this hypothesis and define these regulatory processes at a molecular level. Disruption of ctf1a gene drastically reduces virulence of N. haematococca, but the drastic decrease in cutinase level resulting from this disruption cannot explain the loss of virulence because supplementation of the inoculum with cutinase or mechanical breaching of the cuticle does not recover virulence of the disruptants. Obviously, this Cys6Zn2 binuclear protein (CTF1a) also regulates other gene(s) required for virulence. This project will seek the identity of these virulence genes. The results expected from this project will elucidate novel molecular mechanisms involved in some of the fundamental processes in plant-fungus interaction, possibly suggesting novel approaches to control fungal diseases. Fungal infection causes by far the most damage to food and fiber production. This project could make a significant contributions to an understanding of this interaction that may help manage damaging fungal diseases of plants.