Root-expressed maize lipoxygenase 3 negatively regulates induced systemic resistance to Colletotrichum graminicola in shoots

Abstract

We have previously reported that disruption of a maize root-expressed 9-lipoxygenase (9-LOX) gene, ZmLOX3, results in dramatic increase in resistance to diverse leaf and stalk pathogens. Despite evident economic significance of these findings, the mechanism behind this increased resistance remained elusive. In this study, we found that increased resistance of the lox3-4 mutants is due to constitutive activation of induced systemic resistance (ISR) signaling. We showed that ZmLOX3 lacked expression in leaves in response to anthracnose leaf blight pathogen Colletotrichum graminicola, but was expressed constitutively in the roots, thus, prompting our hypothesis: the roots of lox3-4 mutants are the source of increased resistance in leaves. Supporting this hypothesis, treatment of wild-type plants (WT) with xylem sap of lox3-4 mutant induced resistance to C. graminicola to the levels comparable to those observed in lox3-4 mutant. Moreover, treating mutants with the sap collected from WT plants partially restored the susceptibility to C. graminicola. lox3-4 mutants showed primed defense responses upon infection, which included earlier and greater induction of defense-related PAL and GST genes compared to WT. In addition to the greater expression of the octadecanoid pathway genes, lox3-4 mutant responded earlier and with a greater accumulation of H2O2 in response to C. graminicola infection or treatment with alamethicin. These findings suggest that lox3-4 mutants display constitutive ISR-like signaling. In support of this idea, root colonization by Trichoderma virens strain GV29-8 induced the same level of disease resistance in WT as the treatment with the mutant sap, but had no additional resistance effect in lox3-4 mutant. While treatment with T. virens GV29 strongly and rapidly suppressed ZmLOX3 expression in hydroponically grown WT roots, T. virens Δsml mutant, which is deficient in ISR induction, was unable to suppress expression of ZmLOX3, thus, providing genetic evidence that SM1 function in ISR, at least in part, by suppressing host ZmLOX3 gene. This study and the genetic tools generated herein will allow the identification of the signals regulating the induction of resistance to aboveground attackers by beneficial soil microorganisms in the future.

Keywords: Colletotrichum graminicola; Trichoderma; beneficial microorganisms; hydrogen peroxide; long distance signaling; oxylipin; priming; root-to-shoot signaling.

Figure 1

Colletotrichum graminicola susceptibility factor, ZmLOX3, accumulates preferentially in belowground tissue of maize. (A) Southern corn leaf blight disease symptoms observed under natural field conditions in 2006, College Station, TX, USA of lox3-4 mutant and near-isogenic wild-type maize leaves of the FR2128 background. (B) Semi-quantitative reverse-transcriptase PCR analysis of ZmLOX3 transcript accumulation in C. graminicola infected maize leaves and in untreated roots at 32 and 34 cycles. GAPc at 30 cycles was used as an internal control to normalize cDNA template concentrations. (C) ZmLox3 gene expression from several RNA-seq sources assembled through qTeller. Highlight is expression in leaves (white arrows) comparable with roots (black arrows).

Figure 2

Expression of defense-related genes in leaves of lox3-4 mutant vs B73 NIL (WT) in response to C. graminicola infection. Approximately 3-week-old lox3-4 mutant and WT seedlings at 4 true leaf stage were infected with C. graminicola at 1 × 10⁶ spores.ml⁻¹.mlnicolaola with with at 4 tTrizol reagent and cDNA synthesized with Ambion 1st strand cDNA synthesis kit. PCR was conducted using standard procedures and the expression of GAPc was used as an internal control. HPL, hydroperoxide lyase; PAL, phenylalanine ammonia lyase; GST, glutathione S-transferase; MPI, maize proteinase inhibitor; PR1, pathogenesis related protein 1; GAPc, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Effect of xylem sap from lox3-4 mutant and B73 NIL plants on the pathogenicity of C. graminicola infected on the leaves. The xylem sap were collected from approximately 3-week-old lox3-4 mutant and B73 seedlings at 4 true leaf stage by cutting the stem from the position above the soil. Twenty microliter of sap per plant was injected into the wounding site on the stem of lox3-4 mutant and B73 or TX714 NIL, respectively, subsequently followed by the infection with C. graminicola at 1 × 10⁶ spores/ml. The disease symptoms were recorded and photographed at 3 days post infection on (A) B73 plants treated with H2O or lox3-4 xylem sap, (B) lox3-4 plants treated with H2O or B73 sap, and (C)TX714 plants treated with H2O, B73 sap, or lox3-4 xylem sap. The insert panels show the close-up of representative lesions showing on the corresponding leaf. The (D) lesion size and (E) number of conidia on lox3-4 and B73 leaves infected with C. graminicola upon treatment H2O, and lox3-4 or B73 sap and (F) lesion size on TX714 leaves infected with C. graminicola upon treatment H2O, and lox3-4 or B73 sap. The data are shown as mean ± SE (n > 15 for lesion size and n = 3 for conidia number experiments, respectively). The different letters above the bar indicate the significant difference between different treatments analyzed by SPSS software One-Way ANOVA (p < 0.05).

Figure 4

Effect of colonization of roots of lox3-4 mutant and B73 plants on the pathogenicity of C. graminicola infected on the leaves. Approximately 3-week-old lox3-4 mutant and B73 seedlings at 4 true leaf stage were treated with T. virens in the roots. Then the leaves of treated plants were infected with 10 μ l of C. graminicola conidial suspension at 1 × 10⁶ spores/ml at 3 days post treatment. (A) The disease symptoms were recorded and photographed at 3 days post infection. The insert panels show the close-up of representative lesions showing on the corresponding leaf. (B) The lesion size on lox3-4 and B73 leaves infected with C. graminicola upon treatment with or without T. virens. The data are shown as mean ± SE (n > 15) of lesions examined. (C) The conidia number of C. graminicola from the infected leaves of lox3-4 mutant and WT leaf segment upon treatment with or without T. virens. The data are shown as mean ± SE (n = 3) of leaves examined. The different letters above the bar indicate the significant difference between different treatments analyzed by SPSS software One-Way ANOVA (p < 0.05).

Figure 5

Relative expression of ZmLOX3 in response to root colonization by T. virens strain GV-29-8 and Δ sm1, a mutant that is unable to induce ISR in maize. Two days old disease free seedlings were transferred to hydroponic media with half-strength Murashige and Skooge media and were inoculated with the hyphae of the fungus. At each time point, three biological replicates, each consisting of three plants were sampled. The experiment was repeated twice and results were pooled and analyzed together. The data are shown as mean log₂ (fold change) ± SE and only those means those sample with statistically significant change in gene expression (α = 5%) are designated with an asterisk.

Figure 6

Effect of (A) C. graminicola and (B) alamethincin (ALA) elicitor on accumulation of H2O2 or (C) Nitric Oxide in lox3-4 mutant and B73 leaves. Leaf segment from lox3-4 mutant and B73 (A) inoculated with C. graminicola or (B) treated with ALA for 24 h were stained according to DAB staining procedure to elucidate the ROS accumulation. (C) Nitric oxide accumulation was visualized by DAF fluorecence 4 h after ALA treatment.

Figure 7

Effect of B73 and lox3-4 sap on the expression of defense genes in B73 maize inoculated with C. graminicola. (A) relative expression of SAR-related genes PR1 (pathogenesis related 1), PR5 (pathogenesis related 5), and PAL1 (phennyl alanine amonia lyase), (B) relative expression of genes involved in octadecanoid pathway, AOC (allene oxide synthase), OPR8 (12-oxo-phytodienoic acid 8), LOX5, and LOX10 (lipoxygenase 5 and 10, respectively) and (C) semi-Q-RT-PCR analysis of expression of LOX1, LOX2, LOX9, and OPR7. B73 maize plants at V4 stage were treated with xylem sap from B73 or lox3-4 plants (as described in materials and methods) and were inoculated with C. graminicola 3 h later. Control sample was taken prior to sap treatment and the rest of the samples were taken at designated times after inoculation. For (A) and (B) quantitative real-time PCR (Q-RT-PCR) was used to examine the gene expression and Cullin was used as reference gene. (C) Wherever efficiency of q-RT-PCR reaction was bellow 80% semi-Q-RT-PCR analysis was used to study the gene expression and GAPc was used as internal reference. The data are presented as mean ± SE. The ^†represents the sample that no amplification was detected for the gene expression. The sample with statistically significant change in gene expression (α = 5%) are designated with an asterisk.

Figure 8

Working model of ZmLOX3 involvement in suppression of ISR Responses. In unperturbed maize roots, LOX3-derived oxylipins down-regulate defense-related genes and maintain levels of the defensive and developmental hormones, jasmonate (JA) and ethylene (ET) at healthy levels required for normal seed germination, plant growth, and normal life span (Gao et al., 2008). Upon colonization of roots, Trichoderma virens produces small molecules that possesses signaling activity and modulate gene expression in the roots to allow for mutualistic interactions with the host. One of such signal effecting expression of the host genes is a secreted proteinaceous elicitor, SM1 (Djonovic et al., 2007). One of the functions of SM1-mediated signaling is the down-regulation of expression of the host gene, ZmLOX3, which, in turn, results in de-repression of several defense and signaling host molecules including 9/13-LOX products (such as JA, traumatin, green leaf volatiles, or other oxylipins derived either from C18:3 or C18:2 polyunsaturated fatty acids) and ET. Some of these metabolites, their derivatives or their precursors are transported via xylem into aboveground organs where they signal changes in transcription of a number of defense-related genes including, upregulation of pathogenesis-related genes (e.g., PR5), phenylalanine ammonia lyase (PAL), several defense related LOXs such as LOX1, LOX2, LOX9, and other genes required for biosynthesis of JA, ROS, NO, and cell-wall fortification (e.g., lignin), phytoalexins, and other defense-related metabolites. In addition to up-regulation of defense genes, the host susceptibility genes that promote colonization of tissues by diverse pathogens are down-regulated. This includes LOX10 and LOX5 genes shown to promote disease (Christensen, ; Park, 2011). Collectively, these reprogramming in metabolome and transcriptome results in heightened defense status known as ISR in systemic tissues leading to increased resistance upon pathogen attack.