FUNCTIONAL STUDY OF LIPOXYGENASE-MEDIATED RESISTANCE AGAINST ASPERGILLUS FLAVUS AND FUSARIUM VERTICILLIOIDES INFECTION IN MAIZE

Mycotoxin contamination of maize kernels by fungal pathogens 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 strategies gearing towards hostmediated resistance can be an effective and sustainable approach to strengthen efforts to control the pathogens. Lipid modification into a diverse array of compounds, oxylipins, are recognized in response of plants to fungal pathogens. Lipoxygenase genes (LOXs) play a crucial role in the enzymatic oxidation of polyunsaturated fatty acids (PUFAs) into 9(S)- and 13(S)-hydroperoxides that are further metabolized into different oxylipins including jasmonates, oxo- and keto-fatty acids and volatiles. The involvement of maize LOXs (ZmLOXs) in this respect has been a subject of studies and their genetic manipulation resulted in the alteration of resistance or susceptibility to fungal pathogens in maize. However, the maize genome encodes six 9-LOX and seven 13-LOX isoforms and the specific role of each isoform remains elusive. The current study investigated the role of ZmLOXs in host resistance against the fungi Af and Fv using in silico and in planta approaches. The phylogenetic relationship, sequence similarity, protein domain structures, and transcript level structural variations were explored by comparing publicly available maize pan-genomes. Furthermore, the role of ZmLOXs against Af and Fv infection was investigated through their expression analysis along with further key genes involved in oxylipin biosynthesis, mycotoxin accumulation and lipid profiles in a ZmLOX4 mutant line (UFMulox4) together with W22, Mo17 and Tzi18 inbred lines at 3- and 7-days post-inoculation (dpi). ZmLOX proteins showed considerable variations in their sequences, functional domain structure and transcript structural variations among the pan-genome members. Among the studied lines, Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while the mutant, UFMulox4, was highly susceptible to both pathogens with the most elevated content of mycotoxins. Fv inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to Af . Higher constitutive levels of 9-LOXs genes, ZmLOX1 and ZmLOX2 and an induction of ZmLOX4 were recorded in Tzi18, while the upregulation of ZmLOX1 and ZmLOX4 observed in Mo17 might have larger active role in resistance against Fv. Liquid chromatography-mass spectrometry further revealed an increased accumulation of the 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. Hence, the results confirm the pivotal role of ZmLOXs in controlling the resistance mechanisms against these two pathogens.