Layers of defense responses to Leptosphaeria maculans below the RLM1- and camalexin-dependent resistances

Plants have evolved different defense components to counteract pathogen attacks. The resistance locus resistance to Leptosphaeria maculans 1 (RLM1) is a key factor for Arabidopsis thaliana resistance to L. maculans. The present work aimed to reveal downstream defense responses regulated by RLM1. Quantitative assessment of fungal colonization in the host was carried out using quantitative polymerase chain reaction (qPCR) and GUS expression analyses, to further characterize RLM1 resistance and the role of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) in disease development. Additional assessments of A. thaliana mutants were performed to expand our understanding of this pathosystem. Resistance responses such as lignification and the formation of vascular plugs were found to occur in an RLM1-dependent manner, in contrast to the RLM1-independent increase in reactive oxygen species at the stomata and hydathodes. Analyses of mutants defective in hormone signaling in the camalexin-free rlm1(Ler)pad3 background revealed a significant influence of JA and ET on symptom development and pathogen colonization. The overall results indicate that the defense responses of primary importance induced by RLM1 are all associated with physical barriers, and that responses of secondary importance involve complex cross-talk among SA, JA and ET. Our observations further suggest that ET positively affects fungal colonization

Arabidopsis <i>RabGAP22</i> is required for defense to <i>V. longisporum</i>.

<p>(A) The transcript-derived fragment RR86 from <i>Brassica oleracea</i> accession BRA723 cluster with four Arabidopsis <i>RabGAP</i> genes in maximum likelihood analysis. Bootstrap values >50 are shown. Scale bar represents the number of substitutions per site. (B) Phenotypes of soil-grown plants 28 days post inoculation with <i>V. longisporum</i> showing strong symptoms in <i>rabgap22-1</i> plants (chlorosis, stunting and premature senescence) and only mild symptoms in Col-0. Complementation with the native gene (<i>RabGAP22_Pro:RabGAP22</i>) restored <i>rabgap22-1</i> to the wild-type phenotype. (C) Relative transcript levels of <i>RabGAP11</i>, <i>RabGAP19</i>, <i>RabGAP20</i> and <i>RabGAP22</i> in roots of <i>in vitro</i>-grown Col-0 at 2 d post inoculation with <i>V. longisporum</i>. Data represent means ± SE (n = three pools of >20 plants, repeated twice). (D) Fungal colonization in roots of plants grown in hydroponic culture. Images taken at 7 and 14 d post inoculation with GFP-tagged <i>V. longisporum</i>. (E) Fungal DNA content in roots of plants grown in hydroponic culture at 14 dpi, quantified with qRT-PCR. Data represent means ± SE (n = 3 pools of 5 plants). Asterisk indicates significant difference to Col-0. (Student’s t-test; *p≤0.05; **p≤0.01; ***p≤0.001; ns = not significant).</p

<i>RabGAP22</i> represses jasmonic acid (JA) levels and JA signaling.

<p>(A, B) Endogenous hormone content in <i>in vitro</i>-grown Arabidopsis wild-type and <i>rabgap22-1</i> plants 2 d post inoculation with <i>V. longisporum</i>. (A) JA and (B) JA-Isoleucine (JA-Ile). Data represent means ± SE (n = 3 pools of >50 plants). (C–F) Relative transcript levels of the JA signaling components (C) <i>JAZ10,</i> (D) <i>COI1,</i> (E) <i>VSP2</i> and (F) <i>PDF1.2</i> in roots of <i>in vitro</i>-grown Arabidopsis plants 2 d post inoculation with <i>V. longisporum</i>. Data represent means ± SE (n = 3 pools of >20 plants, repeated twice). Asterisks indicate significant difference to the respective Col-0 mock treated control (Student’s t-test; *p≤0.05; **p≤0.01; ***p≤0.001).</p

Overexpression of <i>LAE1</i> and <i>VEL1</i> represses expression of the polyketide synthase gene, <i>PKS18</i>, associated with melanin production.

<p><b>A.</b> qPCR analyses of <i>PKS18</i>, <i>ChVEL1</i> and <i>ChLAE1</i>. Expression of the genes was determined for the cultures grown for 48 hrs in CM with PGA as the carbon source. WT strain C4, and three independent <i>ChVEL1</i> (left to right: OEVEL1-3, OEVEL1-4 and OEVEL1-7) and <i>ChLAE1</i> (left to right: OELAE1-1, OELAE1-3 and OELAE1-4) overexpression strains each were examined, and expression level relative to the WT sample at 48 hrs is shown. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Single asterisks indicate p-value <0.05, double asterisks indicate p-value <0.001 in T-test analysis in which each strain was compared with WT C4. <i>ChLAE1</i> and <i>ChVEL1</i> are overexpressed in each OE strains and <i>PKS18</i> is repressed in these strains, except for strain OEVEL1-7. The data confirm that ChLae1 and ChVel1 negatively regulate melanin biosynthesis at the transcriptional level. Note that expression of <i>ChLAE1</i> is up in <i>ChVEL1</i> overexpression strains. <b>B.</b> C4, and three independent <i>ChVEL1</i> and <i>ChLAE1</i> (left to right: as above) overexpression strains each, grown as in <b>A</b>. Note that <i>ChVEL1</i> and <i>ChLAE1</i> overexpression strains displayed less pigmentation than the WT C4 strains with <i>ChLAE1</i> OE having the least melanization (compare to <i>Chlae1</i> mutant strains <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002542#ppat-1002542-g007" target="_blank">Figure 7</a>). This indicates that these proteins are negative regulators of melanization of mycelia and that ChLae1 plays a larger role.</p