Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3

Abstract

Oligogalacturonides (OGs) released from plant cell walls by pathogen polygalacturonases induce a variety of host defense responses. Here we show that in Arabidopsis (Arabidopsis thaliana), OGs increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of jasmonate (JA)-, salicylic acid (SA)-, and ethylene (ET)-mediated signaling. Microarray analysis showed that about 50% of the genes regulated by OGs, including genes encoding enzymes involved in secondary metabolism, show a similar change of expression during B. cinerea infection. In particular, expression of PHYTOALEXIN DEFICIENT3 (PAD3) is strongly up-regulated by both OGs and infection independently of SA, JA, and ET. OG treatments do not enhance resistance to B. cinerea in the pad3 mutant or in underinducer after pathogen and stress1, a mutant with severely impaired PAD3 expression in response to OGs. Similarly to OGs, the bacterial flagellin peptide elicitor flg22 also enhanced resistance to B. cinerea in a PAD3-dependent manner, independently of SA, JA, and ET. This work suggests, therefore, that elicitors released from the cell wall during pathogen infection contribute to basal resistance against fungal pathogens through a signaling pathway also activated by pathogen-associated molecular pattern molecules.

Figure 1.

Induction of resistance to B. cinerea by OGs in Arabidopsis plants. A, Lesion development in Arabidopsis Col-0 plants inoculated with B. cinerea 24 h after treatment with a control solution (white circles) or with OGs (black circles). Lesion areas were measured at the indicated times. B, Lesion development in systemic leaves of wild-type plants pretreated with OGs. Lower leaves were infiltrated with OGs (black bars) or water (white bars) and upper, untreated leaves were collected after 72 h and inoculated with B. cinerea. Lesion areas were measured 48 h after inoculation. Values are means ± se of at least 12 lesions. Asterisks indicate statistically significant differences between control and OG-treated plants, according to Student's t test (*, P < 0.05; ***, P < 0.01).

Figure 2.

Induction of resistance to B. cinerea by OGs in mutants impaired in SA, JA, or ET signaling. A, Lesion area in Arabidopsis Col-0 (WT), ein2, and nahG plants treated with a control solution (white bars) or OGs (black bars) and inoculated with B. cinerea 24 h after treatment. Lesion areas were measured 48 h after inoculation. B, Lesion area in Arabidopsis Col-0 (WT), jar1, npr1, and npr1ein2jar1 (nej) plants treated with a control solution (white bars) or OGs (black bars) and inoculated with B. cinerea 24 h after treatment. Lesion areas were measured 48 h after inoculation. C, Lesion area in Arabidopsis Col-0 (WT) or homozygous coi1 plants treated with a control solution (white bars) or OGs (black bars) and inoculated with B. cinerea 24 h after treatment. Lesion areas were measured 48 h after inoculation. Values are means ± se of at least 12 lesions. Asterisks indicate statistically significant differences between control and OG-treated plants, according to Student's t test (*, P < 0.05; ***, P < 0.01).

Figure 3.

Overlap between OG- and fungal infection-induced transcriptional changes. Venn diagram of the number of overlapping and nonoverlapping genes in response to OGs or B. cinerea infection. OG-up, Genes induced 2.0-fold or more (P ≤ 0.01) after 1 or 3 h of treatment with OGs; OG-down, genes repressed 2.0-fold or more (P ≤ 0.01) after 1 or 3 h of treatment with OGs; Bc-up, genes induced 2.0-fold or more (P ≤ 0.01) after 18 or 48 h of inoculation with B. cinerea; Bc-down, genes repressed 2.0-fold or more (P ≤ 0.01) after 18 or 48 h of inoculation with B. cinerea. In parentheses is indicated the total number of genes belonging to each category.

Figure 4.

Expression of genes involved in secondary metabolism in response to OGs and fungal infection. The scheme summarizes the relationships between the shikimate, Trp, Phe, phenylpropanoids, flavonoids, camalexin, indole glucosinolates, and aliphatic glucosinolates biosynthetic pathways and the levels of expression of selected genes in each pathway, portrayed with MapMan software. The number of small squares next to each pathway or portion of pathway indicates how many genes present in the manually compiled list (for details, see Table III) and assigned to that pathway showed a 2.0-fold or greater change of expression (P ≤ 0.01) in response to OGs (A) at 1 h (left squares) or 3 h (right squares) or B. cinerea infection (B) at 18 h (left squares) or 48 h (right squares). Red squares represent genes showing increased expression, blue squares represent genes showing decreased expression. Color intensity indicates the extent of change, expressed as log₂ of the mean ratio between treated and control samples (see scale). Gray dots indicate that none of the genes in the pathway are significantly induced or repressed by the indicated treatment.

Figure 5.

Expression of PAD3 in response to OGs and infection. A, Adult Col-0 plants were sprayed with OGs and total RNA was extracted from rosette leaves harvested at the indicated times (hours). PAD3 expression in each sample was determined by real-time RT-PCR and normalized to the expression of UBQ5. Bars indicate average expression ± sd of two replicates, relative to the expression in untreated Col-0 plants. B, Col-0 (WT), nahG, coi1, npr1, ein2, and jar1 adult plants were inoculated with B. cinerea and total RNA was extracted from inoculated leaves at the indicated times (days post infection). PAD3 expression was determined by RNA-blot analysis. UBQ5 expression confirmed equal loading of the samples (data not shown). C, Col-0 (WT), ein2npr1jar1 (nej), and ups1 seedlings were treated with OGs and total RNA was extracted at the indicated times (hours). PAD3 expression was analyzed by real-time RT-PCR and normalized using the expression of the UBQ5 gene. Bars indicate average expression ± sd of two replicates, relative to the expression in untreated Col-0 plants. D, Col-0 (WT) and coi1 seedlings were treated with OGs and total RNA was extracted at the indicated times (hours). Expression of PAD3 and UBQ5 was analyzed by semiquantitative RT-PCR.

Figure 6.

Induction of resistance to B. cinerea by OGs in wild-type plants and in mutants impaired in camalexin production. Lesion area in Arabidopsis Col-0 (WT), pad3, and ups1 plants treated with a control solution (white bars) or OGs (black bars) and inoculated with B. cinerea 24 h after treatment. Lesion areas were measured 48 h after inoculation. Values are means ± se of at least 12 lesions. Asterisks indicate statistically significant differences between control and OG-treated plants, according to Student's t test (***, P < 0.01). The experiment was repeated three times with similar results.

Figure 7.

Induction of resistance to B. cinerea by flg22. A, Lesion area in Arabidopsis Col-0 (WT), ein2npr1jar1 (nej), and pad3 plants treated with a control solution (white bars) or flg22 (black bars) and inoculated with B. cinerea 24 h after treatment. B, Lesion area in Arabidopsis Col-0 (WT), pad3, and ups1 plants treated with a control solution (white bars) or flg22 (black bars) and inoculated with B. cinerea 24 h after treatment. Lesion areas were measured 48 h after inoculation. Values are means ± se of at least 12 lesions. Asterisks indicate statistically significant differences between control and flg22-treated plants, according to Student's t test (***, P < 0.01). This experiment was repeated twice with similar results.