Genome-wide expression profiling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation

Abstract

Compatibility between plants and obligate biotrophic fungi requires fungal mechanisms for efficiently obtaining nutrients and counteracting plant defenses under conditions that are expected to induce changes in the host transcriptome. A key step in the proliferation of biotrophic fungi is haustorium differentiation. Here we analyzed global gene expression patterns in Arabidopsis thaliana leaves during the formation of haustoria by Golovinomyces cichoracearum. At this time, the endogenous levels of salicylic acid (SA) and jasmonic acid (JA) were found to be enhanced. The responses of wild-type, npr1-1, and jar1-1 plants were used to categorize the sensitivity of gene expression changes to NPR1 and JAR1, which are components of the SA and JA signaling pathways, respectively. We found that the infection process was the major source of variation, with 70 genes identified as having similarly altered expression patterns regardless of plant genotype. In addition, principal component analysis (PCA) identified genes responding both to infection and to lack of functional JAR1 (17 genes) or NPR1 (18 genes), indicating that the JA and SA signaling pathways function as secondary sources of variation. Participation of these genes in the SA or JA pathways had not been described previously. We found that some of these genes may be sensitive to the balance between the SA and JA pathways, representing novel markers for the elucidation of cross-talk points between these signaling cascades. Conserved putative regulatory motifs were found in the promoter regions of each subset of genes. Collectively, our results indicate that gene expression changes in response to infection by obligate biotrophic fungi may support fungal nutrition by promoting alterations in host metabolism. In addition, these studies provide novel markers for the characterization of defense pathways and susceptibility features under this infection condition.

Figure 1.

G. cichoracearum development on susceptible wild-type (wt), npr1-1, and jar1-1 mutant Arabidopsis plants. A, Abundance of haustoria on the plant epidermal cells at 18 and 24 hpi. Percent values are relative to germinated conidia and represent the average ± sd of 15 infected leaves per plant. Similar results were obtained in three independent experiments. B, Bright field images illustrating fungal development at 18 hpi on representative plants of each genotype. Arrows indicate secondary germ tubes, and arrowheads indicate haustoria formed from primary germ tubes. Scale bars, 10 μm. C, Abundance of mature conidiophores per field at 96 hpi assessed by microscopic observation (200×). Values represent the average ± sd of 10 infected leaves per plant. Similar results were obtained in three independent experiments. The asterisk (*) indicates statistically significant differences at P < 0.001.

Figure 2.

Venn diagrams showing the number of Arabidopsis genes differentially regulated during G. cichoracearum haustoria development. The total number of genes identified by each statistical analysis is given in brackets.

Figure 3.

Graphical output of the PCA indicating the spatial ordination of genes influenced by infection and genotype variables. A, Four groups of genes are defined based on their different behaviors in infected wild-type and npr1-1 versus jar1-1 plants (principal component 2 [PC2]), or infected wild-type and jar1-1 versus npr1-1 (principal component 3 [PC3]). Genes are distributed according to fold-change values, indicated in an arbitrary scale. Genes with higher fold-change values are located on the periphery. Genes with distance to the axis intersection <5 were omitted. Infection-mediated repression (R) or induction (I) are indicated on the x and y axes. B, Average fold-change values for each group defined in A are indicated based on their behavior in each plant genotype. Values correspond to average ± sd of T0 versus T18 using the standardized differences of all genes within a cluster. Groups 3 and 4 include genes dependent only on JAR1 or NPR1, respectively.

Figure 4.

Validation of microarray expression data by northern-blot experiments. Total RNA (10 μg/line) isolated from healthy or infected tissues from wild-type (wt), npr1-1 (n), or jar1-1 (j) plants, sampled at the indicated hpi, was analyzed using cDNA probes for the indicated genes. A, Induction (top part) or repression (bottom part) of genes corresponding to the Venn diagram shown in Figure 2 corroborated the results obtained from our microarray assays. B, Expression pattern analysis of genes from Groups 3 and 4, those that could not be induced by infection in jar1-1 or npr1-1 plants (top) and those that were repressed under the same conditions (bottom).

Figure 5.

Northern-blot experiments showing the sensitivity of genes from Groups 3 and 4 to exogenous SA or JA. A and B, RNA was extracted from wild-type plants treated with two different concentrations of SA or JA at 24 h posttreatment (hpt). C, RNA was isolated from wild-type, npr1-1, or jar1-1 plants infiltrated with Pseudomonas syringe pv tomato DC3000 harboring the avrRpm1 gene (5 × 10⁶ cfu/mL; avr) or 10 mm MgCl₂ (mock). The following genes were analyzed by northern blotting: CBS, CBS-domain-containing protein (At5g10860); TURP, tumor-related protein (At3g16640); PGM, phosphoglycerate-mutase-like protein (At3g50520); DI, disulfide isomerase-like protein (At5g18120); ICS1, isochorismate synthase 1 (At1g74710); GH14, glycosyl hydrolase family 14 (At4g17090); F-Box, FBL6/EBF1 F-box-domain-containing protein (At2g25490); AHR, alkyl hydroperoxide-reductase-like protein (At3g06050); MIP, major intrinsic protein aquaporin (At2g36830). Hybridization with rRNA probes was used as a loading control. Probes for the genes PR1 and PDF1.2 were used as controls for the effects of SA and JA.