Lipid communications between Aspergillus flavus and Maize

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843

Cassandra Warren, Eli Borrego, and Michael Kolomiets

Aspergillus flavus is a fungal plant pathogen that infects and contaminates oil-rich crops, such as maize, with mycotoxins contributing to significant economic losses and food safety concerns. Aflatoxin, one of the most potent naturally occurring carcinogenic compounds and its ingestion may result in acute toxicity, hepatic cancer, stunted growth, and immunosupression (Amaike and Keller, 2011). To develop novel disease resistance, it is essential to understand the molecular signaling during the interaction between plants and their parasites. In the midst of this interaction, lipids are important in cell structure and function for both plants and fungi. Oxylipins are a class of oxygenated lipids derived from polyunsaturated fatty acids through oxygenase activity and function as eukaryotic signaling molecules. In fungi, one of the major producers of oxylipins are Psi Producing Oxygenases (Ppo) which incorporate molecular oxygen into fatty acids, namely linoleic acid and oleic acid, to produce precocious sexual inducer (Psi) factors which stimulate asexual and sexual spore development in fungi (Tsitsigiannis and Keller, 2007). The genome of A.flavus contains four Ppo genes which have been characterized to contribute to mycotoxin production, sporulation, and density-dependent development (Brown et al. 2005). In plants, a major contributor of oxylipins production are lipoxygenases (Lox), which incorporate molecular oxygen mainly into linolenic acid at either the 9- or 13- carbon position. Here, oxylipins function to regulate growth and development, senescence, sex determination, defense against biotic and abiotic stress, and programmed cell death (Feussner and Wasternack, 2002). Remarkably, plant and fungal oxylipins are structurally and functionally similar to one another (Tsitsigiannis and Keller, 2006), suggesting molecular mimicry of the host by the pathogen. This has suggested the hypothesis that fungi use host endogenous lipid metabolism enzymes and their oxylipin products to successfully colonize the host, reproduce and synthesize toxins. Previously, 9-LOX disruption alleles, lox3-4 (Gao, 2009), lox5-2, and lox5-3 (Park, unpublished data) were found to be more susceptible to Aspergillus infection than near-isogenic wild-types. When subjected to oxylipin-deficient stains of Aspergillus nidulans, a fungal model and saprophyte, lox3-4 supported increased colonization and sterigmatocystin production compared to wild-type. However, the overlapping roles of both host and parasite oxylipins has not been examined using a conventional fungal pathogen.

Our objective in this study was to elucidate the role of individual maize and fungal oxygenases involved in host-parasite signal-exchange during kernel colonization and subsequent conidiation.

Fig. 4: Conidiation comparison of A.flavus strains between wild-type kernels, lox3-3, lox3-4, lox5-2, and lox5-3 at 3

days post infection. Wild-type and lox3-3 show similar conidiation, lox3-4 and lox5-3 are nearly similar. ΔppoC displays

increased conidiation on 9-LOX mutants, while ΔppoA displays decreased conidiation on 9-LOX mutants; n=4, bars are

means ±SE.

Funding for this project was provided by the Texas A&M University Systems Louis Stokes Alliance for Minority Participation and the National Science Foundation and the College of Agriculture and Life Sciences, Texas A&M University.

Kernel bioassays were performed on maize 9-LOX mutants lox3-3, lox3-4, lox5-2, lox5-3 and wild type line, B73. Genetic stages of these alleles are lox3-3 BC3F3, lox3-4 BC7F5, lox5-2 BC6F7, and lox5-3 BC7F6. Oxylipin-deficient mutants utilized in this study were ΔppoA, C, D, and Δlox (Brown et al., 2009). Kernels were prepared by surface sterilization with 70% ethanol for 5 minutes, sterilized water for 1 minute, 6% sodium hypochlorite for 10 minutes, and three rinses, each for 5 minutes, in sterilized water. To facilitate infection, kernel embryos were wounded with a syringe needle (BD PrecisionGlide 23G1) to a depth of 0.5 cm. 4 kernels were placed into each scintillation vial and their mass was determined. Inoculation consisted of 200 μl per vial of [106 conidia / ml] suspended in 0.01% Tween-20. After vortexing to evenly coat kernels with suspensions, the vials were incubated at 28°C in a humidity chamber under a 12-h-light/12-h-dark photoperiod. After incubation, sporulation was determined as described by Gao et al (2009) with following modifications: 5 ml of 100% methanol were added to each vial and vortexed for 15 seconds, 20 μl of suspension was removed and diluted 1:5 with sterilized water before enumerating conidia with a hemocytometer. For fungal biomass, ergosterol was extracted by grinding kernels from one scintillation vial in 5ml 100% methanol with a blender (Intertek, 700G with 40ml container) for 30s kernels. 10 ml of chloroform was added and the vials were incubated in darkness overnight. Afterwards, 1 ml of supernatant was filtered through a 0.45 μm syringe filter. Ergosterol was quantified by directly injecting a 20 μl aliquot of filtrate into a High Performance Liquid Chromatography (4.6 U ODS-C18 column) with a UV/VIS detection at 282 nm and comparing peak against a standard.

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Fig. 3: Colonization of A. flavus strains on mutant and wild type kernels. Incubated at 28°C for 3 days, 12-h-light/12-h-dark photoperiod

Fig. 1: Comparison of A. flavus wild-type and oxygenase mutants grown on potato dextrose agar used for this study

Introduction

Objective

Experimental Procedure

Figure 1: A. flavus fungal strains

Figure 2: Location of transposon insertions for mutant alleles of ZmLOX3 and ZmLOX5

Fig. 2: Schematic representation of selected maize lipoxygenase gene structure, with transposable element insertion sites designated for mutant alleles. Red boxes represent exon regions, while blue lines represent intron regions.

Figure 3: Kernel bioassay of maize oxylipin mutants exposed to A. flavus oxylipin mutants

Figure 4: Conidiation

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Figure 6: Biomass-dependant conidiation

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Fig. 6: Biomass-dependant conidiation. Wild-type maize and lox3-3 mutant behaved similarly. lox3-4 and lox5-3 behaved nearly similar and displayed increased biomass-dependant conidiation compared to wild-type maize. Δlox and ΔppoC had increased biomass-dependant conidiation on maize 9-LOX mutants compared to wild-type. ΔppoD displayed increased biomass-dependent conidiation on lox3-4 compared to lox5-3; n=4, bars are means ± SE.

Discussion

Acknowledgements

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Fig 5: Ergosterol-based comparison of fungal biomass among selected A.flavus strains between WT kernels, lox3-3, lox3-4, and lox5-3 mutant at 3 days post infection. Wild-type and lox3-3 show similar colonization patterns, while lox3-4 and lox5-2 are again nearly similar. lox3-4 supports less colonization of mutant strains compared to the other genotypes. ΔppoA seems to have a higher colonization rate in lox5-3 but lower in lox3-4 compared to wild-type strain; n=4, bars are means ± SE.

Figure 5: Colonization Abstract

Several species of fungi produce metabolites known as mycotoxins which have negative effects on human and animal

health. Aspergillus flavus threatens maize production by contaminating seed with aflatoxin, a carcinogenic compound

making kernels unfit for human consumption or animal feed. Plant-fungal interactions have been recently hypothesized

to be regulated by numerous lipid signals produced by both the host plant and the pathogen. Growing evidence

suggests that the lipid signals produced by either host plant or pathogen have an effect on the other in the interaction.

Especially highlighted in these interactions are classes of potent lipid signals, known as oxylipins, produced by

Lipoxygenase (LOX) and Psi Producing Oxygenase (Ppo) gene families. While earlier studies have established a

foundation, the exact roles of specific genes remain elusive. Here we aim to uncover this cross-kingdom oxylipin

communication by studying interactions between both oxylipin-deficient mutants of both host and pathogen. In this

study, kernels of maize lipoxygenase mutants lox3-3, lox3-4, lox5-2, and lox5-3 were infected with A.flavus mutant

strains Δlox, ΔppoA, ΔppoC, and ΔppoD as well as the wild type strain, and incubated for 3 and 6 days. Conidiation and

ergosterol analysis are underway for quantifying fungal reproduction and fungal biomass, respectively. This study will

only look at data from 3 days post infection.

KEY WORDS- Aspergillus flavus, maize, lipid signal exchange

•lox3-3 follows patterns very similar to wild-type maize, suggesting that this allele is not a true disruption mutant. This

could be due to the transposon's insertion located in an intron, which may be removed during splicing.

•After conidiation analysis, lox5-2 was removed due to heavy Fusarium spp infection, which could have interfered with

ergosterol analysis.

•lox3-4 and lox5-3 showed similar responses overall in data analysis, which suggests 9-LOX mutants have similar

functions. This could be due to similar oxylipin composition. However, the individual interaction of 9-LOX mutants and

fungal oxygenase mutants are dependent on both the plant and fungal genotypes.

•9-LOX mediated products appear to be involved in promoting colonization and inhibiting sporulation.

•ΔppoA displayed decreased conidiation on 9-LOX mutants. This suggests that a 9-LOX product may compensate for

ΔppoA deficiencies.

•ΔppoC and Δlox displayed increased conidiation on 9-LOX mutants compared to wild-type. This suggests that 9-LOX,

ΔppoC and Δlox products inhibit conidiation.

•ΔppoD displayed increased biomass-dependant conidiation on lox3-4 compared to lox5-3. This suggests that the

lox3-4 product can compensate for ΔppoD, but lox5-3 cannot, revealing that individual plant and fungal oxygenases

have specific roles in host-pathogen interactions.

wild-type Δlox ΔppoA ΔppoC ΔppoD