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

植物角质层是植物与病原真菌相互作用的第一个接触点，是植物最外层的屏障。角质层由不溶性生物聚酯、角质和统称为蜡的可溶性非极性脂质的复杂混合物组成。菌丝体是真菌感染栓必须穿透以感染植物的第一个结构屏障。NSF先前的支持主要负责获得有关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。由入侵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，存在于分生孢子上的少量组成型表达的角质酶从角质释放羟基脂肪酸单体和低聚物，并且这些可溶性产物引起高水平角质酶的诱导，这有助于感染过程。克隆角质酶基因和转录因子，它已被证明，在赤壳菌一个转录因子，CTF 1b，调节组成型表达的角质酶基因（cut 2），和另一个转录因子，CTF 1a，参与角质单体诱导的另一个角质酶基因，cut 1。羟基脂肪酸或其低聚物诱导cut 1的机制仍有待阐明。Cut 1诱导涉及一个回文启动子元件，包含2个重叠的回文。回文2是角质单体诱导cut 1所必需的。回文结合蛋白（PBP）结合cut 1的回文1，并作为一种阻遏物，阻止CTF 1a结合回文2。由于cut 2的回文序列1具有2个核苷酸取代，PBP不能与其结合并抑制cut 2的表达，因此该基因是组成型表达的。据推测，ctf 1a诱导角质单体允许CTF 1a胜过PBP结合cut 1启动子，并导致cut 1诱导。这种角质反应元件结合蛋白（CREBP）的CRE单体或寡聚体刺激的磷酸化使其能够结合CRE并转录激活ctf 1a基因。PBP的磷酸化也可能减弱其与cut 1回文1的结合，从而有助于缓解抑制。本项目将测试这一假设，并在分子水平上定义这些调控过程。ctf 1a基因的破坏显著降低了N.这种破坏导致角质酶水平的急剧下降不能解释毒力的丧失，因为用角质酶补充接种物或机械破坏角质层不能恢复破坏物的毒力。显然，这种Cys 6 Zn 2双核蛋白（CTF 1a）也调节毒力所需的其他基因。本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-真菌相互作用中一些基本过程所涉及的新分子机制，可能为控制真菌病害提供新的方法。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的破坏性真菌疾病。

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.