Loss-of-function mutations in chitin responsive genes show increased susceptibility to the powdery mildew pathogen Erysiphe cichoracearum

Abstract

Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chito-octamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways.

Figure 1.

Chitin localization in the powdery mildew, E. cichoracearum. Confocal images are presented in A to D and transmission electron micrographs in E and F. In A to D, samples were stained with PI (red channel) to highlight fungal structures, although plant structures can be stained with this nonspecific stain, and chitin was localized with the lectin, WGA-Alexa Fluor 488 (green channel). In E and F, WGA-colloidal gold conjugates were used to localize chitin. A, A merged confocal micrograph showing chitin localized to the tip of the appressorium (arrow; 1 dpi). Plant guard cells are also partially stained with PI in this image. Bar = 12 μm. C, Conidium; Ap, appressorium. B, Chitin localization at the growing tip of hypha (arrow) but not on the elongated hyphal region (3 dpi). Hyp, Hyphae. Bar = 11 μm. C, Chitin labeling occurs in the developing conidia still attached to conidiophores (arrows; 7 dpi). Bar = 31 μm. D, A mature conidium (arrow) detaching from a condiophore shows strong chitin localization at the both ends. Bar = 16 μm. E, Chitin occurs in the fungal appressorial cell wall but not on the plant cell wall. CW, Plant outer epidermal cell wall; FCW, fungal cell wall. Bar = 2.15 μm. F, Chitin is found in the haustorial cell wall (arrows). EHMAT, Extrahaustorial matrix, H, haustorium; Nc, haustorial nucleus. Bar = 9.21 μm.

Figure 2.

A, Hierarchical cluster of ratio values (relative to the water control treatment) of 5,435 chito-oligomer responsive genes as determined by the SAM program. Each gene is represented by a single row and each column represents an individual treatment. Magenta represents up-regulated genes; green, down-regulated genes; and black, genes with no change in expression. B, Venn diagrams showing distribution of the subset of these genes showing a ≥1.5-fold change in expression in at least one of the two chito-oligomer treatments relative to the water control. B1, Distribution of 1,252 genes identified as expressed at ≥1.50-fold higher levels in chito-oligomer treated than control samples. B2, Distribution of 1,123 chito-oligomer responsive genes expressed at ≤0.67 of control samples. 8-mer, Treated with chito-octamer; CSC, treated with CSC; Mock, treated with dH2O. Tables with lists of genes in each grouping are presented as supplemental data (Supplemental Tables I–VI).

Figure 3.

Verification of microarrays results for selected genes. A, The top sections present images of the northern blot for each gene. The bottom sections are bar graphs giving the fold-change values for each gene relative to a water control (y axis) for each chito-oligomer treatment (x axis) determined in microarray (white bars) and in northern-blot (black bar) experiments. The ACTIN-2 mRNA (At3g18780) was used as a control in the northern-blot experiments. Genes chosen for northern analysis as follows: AtMPK3, lectin-like protein, ZAT12 and salt-tolerance zinc-finger protein were up-regulated by both the CSC and chito-octamer treatments; the putative auxin-regulated gene was down-regulated by the CSC and chito-octamer treatments and the gene AtERF3 is a gene not regulated by chito-oligomer treatment. B, Transcript levels for At2g35000, encoding an E3 ligase-like gene, and At5g25910, encoding a putative disease resistance protein, at 15 and 30 min after treatment with CSC as determined by RT-PCR. One of three replicates is presented. C, Using the 30-min samples from B, quantitative RT-PCR was used to determine the fold-change in CSC-treated samples relative to water controls. The average and sd for three biological replicates is presented. For comparison, averaged data from the three replicates of the microarray experiment for these genes and the sd are also shown.

Figure 4.

Phenotype of T-DNA mutants. A, Photographs of macroscopic symptoms of leaves inoculated at low density with E. cichoracearum. Leaves of insertional mutants in At2g35000 (SALK_066755) and At5g25910 (SALK_054976) had a slightly higher incidence of macroscopic symptoms as compared with Col-0. All other T-DNA insertional mutants had no change in disease phenotype under low-density inoculums (data not shown). All photos 5 dpi. B, Cleared leaves of infected plants stained with trypan blue. Trypan blue stains the fungal hyphae and conidiophores on the leaf surface. Highest density of fungal growth is seen on insertional mutants of At2g35000 (SALK_066755), At5g25910 (SALK_054976), and transgenic NahG plants. All photographs 5 dpi. Bar = 0.2 mm.