The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

<div><p>Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used <i>Arabidopsis thaliana</i> mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus <i>Botrytis cinerea</i>, which infects a variety of hosts, with the Brassicaceae-specific fungus <i>Alternaria brassicicola</i>. <i>B. cinerea</i> isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while <i>A. brassicicola</i> was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that <i>B. cinerea</i> stimulates the accumulation of glucosinolates to a greater extent than <i>A. brassicicola</i>. In our work with <i>A. brassicicola</i>, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, <i>in vivo</i>, hydrolysis products of indole glucosinolates were found to be involved in defense against <i>B. cinerea</i>, but not in the host response to <i>A. brassicicola</i>. We suggest that the Brassicaceae-specialist <i>A. brassicicola</i> has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist <i>B. cinerea</i> are more sensitive to these phytochemicals.</p></div

Impact of aliphatic glucosinolate on fungal pathogenicity.

<p><i>Arabidopsis</i> leaves from plants containing the double-knockout <i>myb28 myb29</i> (<i>myb28/29</i>) expressed against the Col-0 background (A) and plants in which <i>MYB29^OXP</i> (MYB29) and <i>MYB34^OXP</i> (MYB34) were expressed against the Ler background (B) were inoculated with <i>B. cinerea</i> (B05.10 or grape isolate) or <i>A. brassicicola</i>. Lesion size was measured 72 h after inoculation with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i>. Average lesion sizes from 10 to 17 leaves of each genotype are presented together with the standard errors for each average. All numbers are presented as the relative lesion size as compared to that observed on the corresponding background wild-type plants. Different letters or asterisks above the columns indicate statistically significant differences at <i>P</i>>0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of indole glucosinolate and camalexin on fungal pathogenicity.

<p><i>Arabidopsis</i> mutants <i>cyp79B2/B3</i> and <i>pad3</i>, which have altered total glucosinolate and/or camalexin content, and their corresponding wild-type background (Col-0) were inoculated with <i>B. cinerea</i> (B05.10 or grape isolate) or <i>A. brassicicola</i>. Lesion size was measured 72 h after inoculation (upper and middle panels) with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i> (lower panel). Average lesion sizes from 30 leaves of each genotype are presented along with and the standard error of each average. All numbers are presented as the relative percentage to their corresponding background wild-type. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of glucosinolate-breakdown products on fungal pathogenicity.

<p><i>Arabidopsis</i> mutants with altered total glucosinolate-breakdown product contents and containing different relative amounts of the different type of products were inoculated with the grape isolate of <i>B. cinerea</i> (upper panel), the B05.10 isolate of <i>B. cinerea</i> (middle panel) or <i>A. brassicicola</i> (lower panel). Lesion size was measured 72 h or 120 to 192 h post-inoculation (<i>B. cinerea</i> and <i>A. brassicicola</i>, respectively) on leaves from <i>tgg1-3/tgg2-1</i> (<i>tgg1/2</i>) plants, <i>35S:ESP</i> plants, the wild-types Col-0 and Ler and the triple mutant <i>35:ESP/tgg1-3/tgg2-1</i> (<i>tgg1/2:ESP</i>). (All mutations were expressed against the Col-0 background.) Average lesion areas from 15 to 30 leaves of each genotype are presented together with the standard error of each average. All numbers are presented as the relative lesion size as compared to that observed on the corresponding background wild-type plants. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Glucosinolate accumulation in <i>Arabidopsis</i> after inoculation with a fungal pathogen.

<p>Col-0 <i>Arabidopsis</i> seedlings were inoculated with the B05.10 <i>B. cinerea</i> isolate or <i>A. brassicicola</i> and glucosinolate content was measured 72 h or 120 to 192 h post-inoculation, respectively. GS, glucosinolate. Average glucosinolate accumulation was calculated for 6 to 9 seedlings per treatment and those averages are presented together with their standard errors. Asterisks indicate statistically significant differences relative to the control at <i>P</i><0.05, as indicated by <i>t</i>-tests.</p

Effects of glucosinolate-turnover products on fungal pathogenicity.

<p><i>Arabidopsis</i> leaves from wild-type, <i>pen2</i>, <i>cyp81F2</i> and <i>pen2/cyp81F2</i> plants were inoculated with the grape isolate of <i>B. cinerea</i> (upper panel), the B05.10 <i>B. cinerea</i> isolate (middle panel) or <i>A. brassicicola</i> (lower panel). Lesion size was measured 72 h after inoculation with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i>. Average lesion areas for 30 leaves of each genotype are presented together with the standard error for each average. All numbers are presented as the relative lesion size as compared to the lesions observed on the corresponding background wild-type plants. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Overexpression of <i>AtSHN1/WIN1</i> Provokes Unique Defense Responses

<div><p>The plant cell cuticle serves as the first barrier protecting plants from mechanical injury and invading pathogens. The cuticle can be breached by cutinase-producing pathogens and the degradation products may activate pathogenesis signals in the invading pathogens. Cuticle degradation products may also trigger the plant’s defense responses. <i>Botrytis cinerea</i> is an important plant pathogen, capable of attacking and causing disease in a wide range of plant species. <i>Arabidopsis thaliana shn1-1D</i> is a gain-of-function mutant, which has a modified cuticular lipid composition. We used this mutant to examine the effect of altering the whole-cuticle metabolic pathway on plant responses to <i>B. cinerea</i> attack. Following infection with <i>B. cinerea</i>, the <i>shn1-1D</i> mutant discolored more quickly, accumulated more H₂O₂, and showed accelerated cell death relative to wild-type (WT) plants. Whole transcriptome analysis of <i>B. cinerea</i>-inoculated <i>shn1-1D</i> vs. WT plants revealed marked upregulation of genes associated with senescence, oxidative stress and defense responses on the one hand, and genes involved in the magnitude of defense-response control on the other. We propose that altered cutin monomer content and composition of <i>shn1-1D</i> plants triggers excessive reactive oxygen species accumulation and release which leads to a strong, unique and uncontrollable defense response, resulting in plant sensitivity and death.</p></div

Bacterial proliferation on <i>shn1-1D</i> and WT plants.

<p><b>A,</b><b> </b> Infection phenotypes of representative Ws-0 wild-type and <i>shn1–1D</i> mutant plants at 0–9 days post-inoculation with <i>P. syringae</i> pv. <i>tomato</i> DC3000. <b>B,</b> Quantitative analysis of bacterial growth in WT and <i>shn1–1D</i> mutant plants is presented. Results represent means±SE (n = 6). Asterisk denotes statistical difference between WT and <i>shn1–1D</i> plants calculated for the specified time point by Student’s t-test (<i>P</i><0.05).</p

Effect of cutin monomers on disease symptoms and gene expression.

<p><b>A,</b> WT leaves were inoculated with <i>B. cinerea</i> spores supplemented with 0.04, 0.4 or 0.8 µg/cm² cutin monomers extracted from either <i>shn1–1D</i> (<i>shn1–1D</i>-CM) or the WT (WT-CM). As a control, we used WT and <i>shn1-1D</i> leaves inoculated with <i>B. cinerea</i> only. Presented are means±SD of chlorotic area of 15 leaves 72 h post-inoculation. Different letters represent significant difference by Tukey-Kramer HSD analysis (<i>P</i><0.05). <b>B,</b> Expression of selected genes in WT leaves inoculated with <i>B. cinerea</i> spores supplemented with 0.04 µg/cm² cutin monomers extracted from either <i>shn1–1D</i> (WT+<i>shn1–1D</i>-CM) or WT (WT+WT-CM). As a control, we used WT and <i>shn1–1D</i> leaves inoculated with <i>B. cinerea</i> only. <b>C,</b> PAL1 expression in WT leaves supplemented with 0.04 µg/cm² cutin monomers extracted from either <i>shn1–1D</i> (WT+<i>shn1–1D</i> CM) or WT (WT+WT-CM).</p

Differential gene regulation by <i>B. cinerea</i> in <i>shn1-1D</i> and WT.

<p><b>A,</b> Venn diagram representing overlapping or non-overlapping gene sets differentially expressed in WT or <i>shn1–1D</i> plants 72 h after infection with <i>B. cinerea</i> and defined by FC >2 (<i>P</i><0.05). <b>B,</b> Relative gene expression between inoculated and noninoculated <i>shn1–1D</i> and WT plants. Expression of selected genes from microarray data validated using qRT-PCR on cDNA extracted from <i>shn1–1D</i> or WT leaves 72 h after inoculation with <i>B. cinerea</i> relative to noninoculated leaves (mock).</p