Establishment of maize resistance to fungal diseases by host-induced gene silencing and site-directed mutagenesis

Abstract

Maize is one of the most cultivated crops in the world. A disease called anthracnose accounts for up to 80% of the loss in maize production. It is caused by the hemibiotrophic fungus Colletotrichum graminicola. Unfortunately, the disease is notoriously difficult to combat, since host resistance mechanisms are hardly available. In the present investigation, the principle of host-induced gene silencing (HIGS) was employed to protect maize plants from C. graminicola infection. HIGS is an RNA-interefence (RNAi)-based process, wherein plant-produced short interfering RNAs (siRNA) are taken up by the fungus and trigger the silencing of cognate genes of the latter. In the present study, genes encoding fungicide targets were chosen as HIGS targets, namely C. graminicola -Tubulin 2 and Succinate dehydrogenase 1. RNAi vectors were designed using appropriate regions of these target genes. Transgenic plants expressing RNAi constructs were infected with C. graminicola, whereby the plants showed quantitative resistance. In addition to the HIGS approach, a further strategy was pursued, which consisted in knocking out a susceptibility factor against C. graminicola by means of targeted mutagenesis. This factor was the 9-LIPOXYGENASE LOX3 gene from maize, for which several mutated plants were generated by expression of RNA-directed Cas9 endonuclease. Homozygous lox3 mutants were tested in C. graminicola infection assays to analyze the consequences of their mutations. Quantification of fungal biomass revealed that the lox3 mutants were significantly less colonized by C. graminicola compared to the non-mutated wild-type. Corn common smut, another important fungal disease, is caused by the biotrophic pathogen Ustilago maydis. Transcriptional data (Doehlemann et al., 2008) collected during the course of infection with U. maydis showed that, depending on the infection, several members of the LOX gene family are upregulated, one of which is LOX3. Therefore, the available lox3 mutants were tested for their response to infection with U. maydis. The quantification of the disease symptoms showed that the lox3 mutants showed a moderate resistance against U. maydis infections. Furthermore, the quantification of the biomass of U. maydis revealed that the lox3 mutants were colonized by the fungus to a lesser extent compared to the wild-type. Furthermore, infection tests were performed using lox3 mutants independently produced by transposon insertion mutagenesis. These lines showed a resistance behavior similar to that of Cas9-induced mutants, by which the anticipated role of LOX3 for the interaction of maize and U. maydis was corroborated. From the literature it is known that U. maydis suppresses the accumulation of reactive oxygen species (ROS) to establish its biotrophic mode of pathogenesis. A ROS accumulation test revealed that lox3 mutants feature increased ROS accumulationcompared to the wild-type, suggesting that the immunity of the mutants triggered by pathogen-associated molecular pattern (PAMP) led to a reduction in the severity of fungal infection. This is the first study showing that lox3 mutants show moderate resistance to U. maydis. In view of these results, it is concluded that LOX3 is a susceptibility factor for U. maydis as well