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

植物角质层是植物与真菌病原菌相互作用的第一个接触点，是植物最外层的屏障。角质层由一种不溶的生物聚合体、角质和一种复杂的可溶性非极性脂混合物组成，统称为蜡质。角质是真菌侵染植物必须穿透的第一道结构性屏障。NSF先前的支持主要负责获得关于角质的化学和生物合成的基本信息，以及角质层在植物-真菌相互作用中的作用。入侵的真菌分泌的角质酶帮助它附着在宿主上并穿透外部屏障。根据先前获得的证据，当真菌分生孢子接触植物表面时，分生孢子上存在的少量结构性表达的角质酶从角质中释放出羟基脂肪酸单体和寡聚体，这些可溶性产物导致诱导高水平的角质酶，从而帮助感染过程。通过克隆角质酶基因和转录因子，已经证明在红球线虫中，一个转录因子CTF1b调节一个结构性表达的角质酶基因(Ut2)，另一个转录因子CTF1a参与另一个角质酶基因ut1的角质单体诱导。羟基脂肪酸或其寡聚体诱导KAT1的机制尚不清楚。Cut1的诱导涉及一个回文启动子元件，它包含2个重叠的回文序列。角质单体对Cut1的诱导需要回文2。回文结合蛋白(PBP)能结合ut1的回文1，并抑制CTF1a与回文2的结合。由于ut2的回文1有2个核苷酸替换，因此PBP不能与其结合并抑制ut2的表达，因此该基因是结构性表达的。推测角蛋白单体对CTF1a的诱导作用使CTF1a与PBP竞争，与Cut1启动子结合，导致Cut1的诱导。角质反应元件结合蛋白(CREBP)的角质单体或寡聚体刺激的磷酸化使其与Cre结合并在转录上激活ctf1a基因。也有可能PBP的磷酸化可能会削弱其与ut1的回文1的结合，从而帮助缓解这种抑制。该项目将检验这一假设，并在分子水平上定义这些调控过程。Ctf1a基因的破坏大大降低了红球菌的毒力，但这种破坏导致的角质酶水平的急剧下降并不能解释毒力的丧失，因为接种角质酶或机械破坏角质层不能恢复干扰物的毒力。显然，这个Cys6Zn2双核蛋白(CTF1a)还调节着毒力所需的其他基因(S)。该项目将寻求这些毒力基因的身份。该项目的预期结果将阐明植物-真菌相互作用的一些基本过程中涉及的新的分子机制，可能会为控制真菌疾病提供新的方法。到目前为止，真菌感染对食物和纤维生产造成的损害最大。该项目可以为理解这种相互作用做出重大贡献，这可能有助于管理植物破坏性的真菌疾病。

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.