Functional Identification Of Cell Wall Biosynthesis-related Genes And Their RNAi Molecules In Fusarium Graminearum

Fusarium graminearum(Fg) is an ascomycete that causes Fusarium head blight(FHB) in wheat, maize and other small grain cereal crops worldwide. Recently, global climate change has aggravated the spread and severity of FHB to even wider regions, therefore caused huger yield losses. In addition, Fusarium graminearum produces various mycotoxins during infection that accumulate in the grain, posing a serious threat to human and animal health. The pathogen infects a wide range host, not only members of the Gramineae, but also other plant species without causing obvious disease symptoms. Using chemical fungicides as a method to control FHB has dramatically increased the emergence of fungicide-resistant FHB pathogens and is harmful to the environment. Using of biocontrol agents is another method to control FHB. However, biocontrol methods lack successful large-scale applications since the disadvantages of low viability and delivery mechanisms. Thus, it is necessary to investigate new method that can efficiently control FHB in wheat.RNA interference(RNAi) is a conserved mechanism in eukaryote that offers opportunities to generate new traits in improvement of crop disease resistance. Fusarium cell wall is the first structure during the initial association with host. With a function of avoiding damage by external environment, the cell wall is also very important for the cell morphogenesis and pathogenicity in Fusarium. In our resesrches, cell wall biosynthesis-related genes were selected and their functions for pathogenicity and development were well studied. There are four elements in this thesis: functional analysis of trehalose synthesis genes TPS1 and TPS2; establishment of RNA interference system in Fusarium; functional analysis and high efficiency fragments selection of chitin synthase gene Chs3 b gene; functional analysis of glyoxal oxidase gene GLX that recognized by disease-resistance antibody CWP2. The main results were as follows:1. TPS1 and TPS2 were involved in trehalose biosynthesis in F. graminearum. Five genetically different Fusarium strains were generate: Δtps1, Δtps2 and Δtps1-Δtps2, each carried either a single gene disruption or double disruption genes; two complementation strains, TPS1 C and TPS2 C, were generated by reintroducing the TPS1 gene into an ectopic locus of the Δtps1 mutant or the TPS2 gene into the Δtps2 mutant, respectively. The three null mutants Δtps1, Δtps2 and Δtps1-Δtps2 simultaneously lost their abilities to synthesize trehalose. There was no significant difference in development and pathogenicity of Δtps1 and Δtps1-Δtps2 compared with the WT Fg5035, however, the mycotoxin biosynthesis was reduced by 67% and 60% in Δtps1 and Δtps1-Δtps2, respectively. The Δtps2 mutant strain showed extremely slow growth and barely formed aerial hyphae; in addition, it abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution. Deletion of TPS2 led to a 99% drop in virulence and a 86% drop in mycotoxins production compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively.2. A series of RNAi vector were constructed for single gene interference, double genes interference, gene high-efficiency interference and Gateway RNAi vector rapid construction, respectively. p RNAi was the single gene RNAi vector that contained Sma I and Sac II restriction site using for hairpin structure jointing; p RNAi-linker was the double genes RNAi vector that contained Sma I/Sca I and Sac II/Swa I restriction site used for the jointing of hairpin structures from two different genes; p RNAi-Dpromoter which contained two strong promoters without terminator was constructed for gene high-efficiency interference. Gene hairpin structure was inserted into p RNAi-Dpromoter between Sma I and Sac II restriction site. The Gateway RNAi vectors included two vectors, donor vector p DON201 and entry vector p SXSH-gateway D which had four att L regions that used for gene hairpin structure construction from a sense fragement.3. Fusarium graminearum contains eight Chs genes. Among the eight Chs members, the Chs3 b gene expressed at the highest levels during Fg colonization on wheat spikes. To generate RNAi constructs with-500 bp, the Chs3 b gene was divided into five segments(15-583 nt, 570-1149 nt, 1130-1629 nt, 1612-2134 nt, 2104-2685 nt) to make five different RNAi constructs(named Chs3 b RNAi-1, Chs3 b RNAi-2, Chs3 b RNAi-3, Chs3 b RNAi-4 and Chs3 b RNAi-5). All the five segments were successful to interferer the expression level of Chs3 b m RNA. Among the five segments, Chs3 b RNAi-1,-3 and-5 were the highest three segments to interferer the Chs3 b gene. Retarded growth for these three strains was observed on PDA media. Chs3 b RNAi-1 lost the ability to produce ascospore and had a 50% reduction in sporulation. Chs3 b RNAi-2 had a 64% reduction in sporulation. Microscopic analysis revealed that strains Chs3 b RNAi-1 and-5 had clearly restricted, severely distorted, and crooked mycelia and increased width and conglobated structures of conidia. Correspondingly, the Chitin contents of Chs3 b RNAi-2,-3, and-5 decreased significantly. Whereas a 9.5% significance of Chitin content increase was observed for strain Chs3 b RNAi-4 compared to the nontransgenic strain. The virulence of the transgenic Fg strains was reduced significantly except strain Chs3 b RNAi-4 when assayed by seedling inoculation and single-floret injection of two wheat varieties. Moreover, no off-target sequences were found in wheat and/or other organisms for the Chs3 b gene and the si RNA could enter into Fusarium cell during conidispore germination or Fusarium protoplast.4. Westernblots revealed that a glyoxal oxidase(Glx) is the antigen of CWP2. This Glx efficiently catalyzed H2O2 production, and this enzymatic activity was specifically inhibited by the CWP2 antibody. Cellular localization studies showed that Glx was located to the plasma membrane. GLX-deletion strains of Fg, F. verticilloides, and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was down-regulated.Our studies revealed three cell wall biosynthesis-related genes were required for development and virulence of Fusarium graminearum: Chs3 b gene and TPS2 gene were both important in Fusarium graminearum development and virulence; GLX was related to virlence and mycotoxin biosynthesis in Fusarium. On the other hand, a high efficiency RNA interference system was established in Fusarium graminearum and three RNAi fragments Chs3b-1,-3, and-5, were selected for the strongest overall interfering effects on mycelium development and could be used as interference candidate fragments.