Abstract
Biotic stresses caused by microbial pathogens impair crop yield and quality if not restricted by expensive and often ecologically problematic pesticides. For a sustainable agriculture of tomorrow, breeding or engineering of pathogen-resistant crop varieties is therefore a major cornerstone. Maize is one of the four most important cereal crops in the world. The biotrophic fungal pathogen Ustilago maydis causes galls on all aerial parts of the maize plant. Biotrophic pathogens like U. maydis co-evolved with their host plant and depend during their life cycle on successful manipulation of the host’s cellular machinery. Therefore, removing or altering plant susceptibility genes is an effective and usually durable way to obtain resistance in plants. Transcriptional time course experiments in U. maydis-infected maize revealed numerous maize genes being upregulated upon establishment of biotrophy. Among these genes is the maize LIPOXYGENASE 3 (LOX3) previously shown to be a susceptibility factor for other fungal genera as well. Aiming to engineer durable resistance in maize against U. maydis and possibly other pathogens, we took a Cas endonuclease technology approach to generate loss of function mutations in LOX3. lox3 maize mutant plants react with an enhanced PAMP-triggered ROS burst implicating an enhanced defense response. Based on visual assessment of disease symptoms and quantification of relative fungal biomass, homozygous lox3 mutant plants exposed to U. maydis show significantly decreased susceptibility. U. maydis infection assays using a transposon mutant lox3 maize line further substantiated that LOX3 is a susceptibility factor for this important maize pathogen.
Highlights
Maize (Zea mays L.) is one of the most important cereal crops in the world
From a total of 140 Agrobacterium-infected Hi-II A x B immature embryos, 88 putative primary transgenic plants were generated, all of which were proven by transgene-specific PCR analyses for clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 endonuclease, guide RNA (gRNA) and hygromycin phosphotransferase to carry the T-DNA derived from the transformation vector pNB104 (Figure 1B)
Lox3 mutants were engineered by Cas9 endonuclease technology
Summary
Maize (Zea mays L.) is one of the most important cereal crops in the world. As a fast growing C4 plant, its kernels are used for direct human consumption, its biomass for feed and biofuel production, and it is a source of raw material for the chemical and food industries (Pathi et al, 2013). In the context of a growing world population and food demand, there is an urgent requirement to develop crop varieties with broad-spectrum resistance (Dangl et al, 2013). Plants co-evolved with the selection pressure of invading pathogens, leading to a sophisticated, multilayered, and interconnected innate immune system. Intracellular signaling events stimulate the production of reactive oxygen species (ROS) in the extracellular space and intracellularly a transcriptional reprogramming of the plant. This reaction is called PAMPtriggered immunity (PTI) which strengthens defense, for example, by the secretion of antimicrobial peptides/compounds (Saijo et al, 2018). Build-in growth-defense antagonisms in the plant metabolism and hormone signaling are exploited to suppress specific defense pathways depending on the lifestyle and the specific requirements of the pathogen
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