Priming of wheat with the green leaf volatile Z-3-hexenyl acetate enhances defense against Fusarium graminearum but boosts deoxynivalenol production

Abstract

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection.

Figure 1.

Preexposure with Z-3-HAC leads to lower fungal biomass, smaller necrotic lesions, and higher DON content in wheat seedlings. A, Percentage of spikelets (n = 27) showing necrotic lesions after pretreatment with Z-3-HAC at 0, 2 4, 6, and 8 d after infection (DAI). Significant differences between treatments are depicted with asterisks. Significance was determined using the χ² test with a significance level of 0.05. B, Leaves of seedlings preexposed to Z-3-HAC or MeSA show smaller necrotic lesions compared with nonprimed control seedlings, while preexposure to MeJA exacerbates lesion length. Leaves were cut from the seedlings and subsequently wounded, after which a droplet of a conidia suspension of F. graminearum (5 × 10⁴ conidia mL⁻¹) was applied on the wound. Lesion length was monitored at 24, 48, and 72 hai. Bars represent means of 15 biological replicates. Bars depicted with different letters per time point indicate significant differences between the treatments (P < 0.05). Error bars represent se. C, Photographs depicting representative necrosis symptoms at 72 hai. The top row shows leaves that have been primed with Z-3-HAC, while the bottom row shows leaves that have not been primed. D, Normalized quantitative relative values (NRQ) of fungal biomass. Leaf sheaths were exposed overnight to Z-3-HAC. The next day, a conidia suspension of F. graminearum (5 × 10⁵ conidia mL⁻¹) was applied in the leaf sheaths. Biomass was determined using pre-mRNA slicing factor of F. graminearum (FGSG_01244) as a reference gene and expressed relative to the plant reference genes cell division control protein (Ta54227) and protein transport protein Sec23A (Ta35284). Bars represent means of two biological repeats of four pooled leaf sheaths each. E and F, Microscopic images illustrate the formation of infection structures at 24 hai (E) followed by invasion of the plant cell (F).

Figure 2.

Expression profiles of PAL, ICS, LOX1, and LOX2 at 24 and 48 h after challenge with a conidia suspension of F. graminearum. Data represent means of four biological replicates, each consisting of four pooled leaf sheaths. Error bars represent se. Significant differences between the treatments per time point (P < 0.05) are depicted with asterisks.

Figure 3.

Leaves of seedlings preexposed to Z-3-HAC and treated with MeJA at 24 hai show smaller lesions compared with the control treatment. Leaves were cut from the seedlings and subsequently wounded, after which a droplet of a conidia suspension of F. graminearum (5 × 10⁴ conidia mL⁻¹) was applied on the wound. Lesion length was monitored at 24, 48, and 72 hai. Arrows indicate the time points at which the seedlings were treated with 10 µL of MeJA applied on a filter paper. Bars represent means of 10 to 15 biological replicates. Bars depicted with different letters per time point indicate significant differences between the treatments (P < 0.05). Error bars represent se.

Figure 4.

Expression profiles of PR1, PR4, PR5, and PEROX at 24 and 48 h after challenge with MeSA or MeJA. Data represent means of three biological replicates, each consisting of four pooled leaf sheaths. Error bars represent se. Different letters per time point indicate significant differences between the treatments (P < 0.05).

Figure 5.

Expression profiles of PAL, ICS, LOX1, LOX2, PR1, PR4, PR5, and PEROX at 24 and 48 h after challenge with a conidia suspension of F. graminearum. Data represent means of four biological replicates, each consisting of four pooled leaf sheaths. Error bars represent se. Different letters per time point indicate significant differences between the treatments (P < 0.05).

Figure 6.

DON concentrations (pg mg⁻¹ plant dry weight) at 24 and 48 h after challenge with a conidia suspension of F. graminearum. Data represent means of four biological replicates, each consisting of six to eight pooled leaf sheaths. Error bars represent se. A significant difference between treatments per time point (P < 0.05) is depicted with an asterisk.