Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

Abstract

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.

Keywords: Colletotrichum acutatum; Fragaria × ananassa; quantification of gene expression; salicylic and jasmonic acid; strawberry defense response.

Figure 1

Colletotrichum acutatum development in strawberry tissue. (A) Progression of C. acutatum isolate CECT 20240 conidial germination. (B–F) Photomicrographs of C. acutatum structures formed during infection of strawberry leaves over a 9-days period. (B) Non germinated conidia at 1 dpi; (C) Germinated conidia after 3 dpi; (D,E) Appressorium and mycelium development at 5 and 7 dpi, respectively; (F) Abundant mycelium at 9 dpi. All bars 25 μm.

Figure 2

Transmission Electronic Microscopy (TEM) in strawberry crown. (A) Penetration of cuticle and biotrophic stage of C. acutatum by subcuticular hyphae on wall epidermal cell 36 h post-inoculation. (B) Necrotrophic signals of development of C. acutatum by intercellular hypha 4 days post-inoculation. (C) Necrotrophic development of C. acutatum by intracellular hyphae in xylem tissues at 7 dpi. A, appresorium; C, plant cuticle; IV, infection vesicle; W, wall plant cell; IM, intramural o intercellular hypha; FW, fungal cell wall; CD, cell debris; IC, intracellular hyphae; X, xylem. Bar = 1 μm.

Figure 3

Light microscopic analysis of DAB stained preparations of C. acutatum infected strawberry tissue at 7 dpi, and relative expression values by RTqPCR analysis of FaGST1 (At1g02930 ortholog) gene at different time points of infection. (A) Tissue sample showing no DAB reaction, (B,C) very rarely detected reduction of DAB beneath fungal appressoria, and surrounding the penetration pegs, at upper and lower focal planes, respectively. All bars 25 μm. (D–F) Relative FaGST1 expression values. At each time point, every inoculated sample was compared with its corresponding mock treated sample. In the graphics, standard value 1 at T0 was added to better illustrate changes.

Figure 4

Singular Enrichment Analysis (SEA) on strawberry up-regulated genes. FatiGO was used to extract relevant GO terms for biological processes (BP) at p < 0.005. The terms are considered to be relevant by the application of statistical tests as described in Al-Shahrour et al. (2004). Data are presented as a heat map, as prompts color intensity correlates with adj. p-value, the highest intensity, the lowest adj. p-value. See Table S1 for a detailed list of further genes belonging to overrepresented functions.

Figure 5

Relative expression values by RTqPCR analysis of upregulated strawberry genes during C. acutatum infection. (A) Relevant strawberry genes in this study, (B) JA-responsive marker genes, and (C) SA-responsive marker genes. Strawberry crowns were harvested at different days post-treatment (dpi) either with mock or C. acutatum spore suspension. At each time point, every inoculated sample was compared with its corresponding mock treated sample. In the graphics, standard value 1 at T0 was added to better illustrate changes. Asterisk marks genes not present in the Array dataset. Arabidopsis orthologous genes are AT5G13080 (AtWRKY75), AT5G42650 (AtAOS), AT2G38470 (AtWRKY33), AT3G45140 (AtLOX2), AT2G46370 (AtJAR1), AT5G44420 (AtPDF1.2), AT3G48090 (AtEDS1), AT3G52430 (AtPAD4), AT1G28480 (AtGRX480), AT3G56400 (AtWRKY70), AT2G14610 (AtPR1), AT3G57260 (AtPR2).

Figure 6

Relative expression values by RTqPCR analysis of upregulated strawberry genes by hormone treatment. (A) Relevant strawberry genes in this study, (B) JA-responsive marker genes, and (C) SA-responsive marker genes. Strawberry plantlets were treated with mock, SA (5 mM) and MeJA (2 mM) elicitors, and harvested at different hours post-treatment (htp). At each time point, every elicited sample was compared against its corresponding mock treated sample. Left and right legends represent relative expression values for SA and JA treatments, respectively. In the graphics, standard value 1 at T0 was added to better illustrate changes. Asterisk marks genes not present in the Array dataset. Arabidopsis orthologous are AT5G13080 (AtWRKY75), AT5G42650 (AtAOS), AT2G38470 (AtWRKY33), AT3G45140 (AtLOX2), AT2G46370 (AtJAR1), AT5G44420 (AtPDF1.2), AT3G48090 (AtEDS1), AT3G52430 (AtPAD4), AT1G28480 (AtGRX480), AT3G56400 (AtWRKY70), AT2G14610 (AtPR1), AT3G57260 (AtPR2).

Figure 7

Quantification of (A) SA and (B) JA [ng g-1 (dry weigh)] in aerial tissues of in-vitro strawberry plantlets at 3 and 5 days after mock (white bars) and pathogen inoculation (black bars). Data are the mean of three biological replicates, and error bars represent the SD. Numbers inside arrows indicate percentage of increase of infected samples vs. mock. Asterisks indicate confidence of ANOVA-Bonferroni Multiple Comparisons Test (^***p < 0.001; ^**p < 0.01).

Figure 8

Hypothetical model of SA- and JA-dependent defense pathways activated in strawberry in response to C. acutatum. This model is based on the canonical pathways described in model plant. Upon interaction with C. acutatum, the strawberry plant activates upstream components of SA and JA defense pathways. Thus, synthesis of these signal molecules increases and main downstream key components for SA (FaNPR1, FaWRKY70, FaGRX) and JA (FaWRKY33) are activated. Unlike Arabidopsis WRKY33, FaWRKY33 does not act as a negative regulator of the entire SA-dependent defense signaling pathway, either by a direct or an indirect effect of fungal activity, but only for some components (FaGST1, FaPR1.1). That allows FaGRX, together with FaNPR1 and FaWRKY70, to act as negative regulators of JA responsive genes similarly to their Arabidopsis orthologs. As a result, important JA-responsive defense marker genes, such as FaLOX2, FaJAR1, and FaPDF1, are not induced. These impaired mechanisms might provide some advantage for fungal spreading.