Functional Study of Lipoxygenase-mediated Resistance to Fungal Pathogens in Maize for Milk-chain Safety and in Grapevine for Sustainable Viticulture

Fungal pathogens present a big concern in grapevine and maize production worldwide. Powdery mildew, a disease caused by an obligate biotroph, Erysiphe necator (En) is the most important fungal disease affecting grapevines, whereas. mycotoxin contamination of maize by Aspergillus flavus (Af) and Fusarium verticillioides (Fv) is a chronic global challenge impacting food security, health and trade. Current disease management practices are proven inadequate and host-mediated resistance can be an effective approach to control these pathogens. Lipid membrane modification and peroxidation of polyunsaturated fatty acids (PUFAs) into oxylipins are reported in plants as a response to fungal pathogens. In this respect, lipoxygenase genes (LOXs) play a crucial role in the peroxidation of polyunsaturated fatty acids (PUFAs) into 9(S)- and 13(S)-hydroperoxides, which are further metabolized to oxylipins, including jasmonates, oxo- and keto-fatty acids and volatiles. However, grapevine and maize genome each encodes 13 lipoxygenase isoforms and the specific role of each isoform in plant-pathogen interaction remains elusive. Hence, the PhD project was aimed at identifying and characterizing lipoxygenase isoforms involved in these metabolic and signalling pathways. Grapevine and maize genotypes with a resistant and susceptible background to fungal pathogens were used in controlled infection experiments to assess the LOX expression pattern, peroxidation and phytohormone profile, and lipid modification. Subsequently, pertinently modulated grapevine LOX isoforms were modified in planta with overexpression or CRISPR/Cas9 mediated knock-out strategies for in-depth functional analysis. In maize, expression analysis of ZmLOX isoforms and key genes involved in oxylipin biosynthesis, mycotoxin accumulation and lipid profiles were studied in a ZmLOX4 mutant line (UFMulox4) along with W22, Mo17 and Tzi18 inbred lines. Tzi18 showed the highest resistance to both pathogens with significantly lower mycotoxin accumulation, while UFMulox4 was highly susceptible. Fv inoculation resulted in a stronger induction of ZmLOXs as compared to Af infection and higher constitutive levels of 9-LOX genes, ZmLOX1 and ZmLOX2, as well as the induction of ZmLOX4 were recorded in Tzi18 and Mo17. Oxylipin profiling using liquid chromatography-mass spectrometry (LC-MS) revealed an increased accumulation of linoleic (18:2) derived 9-cyclopentenone, 10-oxo-11-phytoenoic acid (10-OPEA), in Fv inoculated kernels of Tzi18 and Mo17, which was previously identified to inhibit fungal growth in vitro. As regards grapevine, a controlled infection experiment with En was performed using susceptible (Teroldego) and resistant (NY_39) genotypes, and gene expression patterns were analyzed in inoculated (12, 24 and 48hpi) and control leaves. En triggered a wide range of differential changes in the transcriptional response of VviLOX isoforms and JA and SA-related genes, lipidomic and phytohormonal profiles between the genotypes. Stable Agrobacterium-mediated gene knockout (through CRISPR/Cas9) of VviLOXO and 7, and overexpression of VviLOX13 were performed on embryogenic calli of V. vinifera cv. Sugraone-a genotype susceptible to En- for in-depth analysis of the specific role of these VviLOXs in plant-pathogen interaction. Vviloxo and Vvilox7 knock-out lines produced morphologically altered plants with reduced leaf size, internode length, plant height and lower chlorophyll production. Moreover, knock-out lines showed different lipid and phytohormonal profiles as compared to wild-type (WT) plants. Both types of knock-out lines exhibited higher resistance than WT upon inoculation of En and Botrytis cinerea (Bc) in vitro. Mutant lines overexpressing VviLOX13 showed no aberrations in growth and development, while lipidomic and phytohormonal analysis showed distinct differences in several lipidomic classes and increased accumulation of abscisic acid. In planta and in vitro infection assays with En and Bc did not show a different behaviour in the transgenic plants compared to the WT control, which was highly susceptible.