TaMAPK4 Acts as a Positive Regulator in Defense of Wheat Stripe-Rust Infection

Abstract

Highly conserved mitogen-activated protein kinase (MAPK) cascades regulate numerous plant processes, including hormonal responses, stress, and innate immunity. In this research, TaMAPK4 was predicted to be a target of tae-miR164. We verified the binding and suppression of TaMAPK4 by co-expression in Nicotiana benthamiana. Moreover, we found TaMAPK4 was localized in the cytoplasm and nucleus using transient expression analyses. TaMAPK4 transcripts increased following salicylic acid (SA) treatment and when host plants were infected with an avirulent race of the stripe-rust pathogen. Silencing of TaMAPK4 by virus-induced gene silencing permitted increased colonization by the avirulent pathogen race. Detailed histological results showed increased Puccinia striiformis (Pst) hyphal length, hyphal branches, and infection uredinial size compared to the non-silenced control. SA accumulation and the transcript levels of TaPR1, TaPR2, and TaPR5 were significantly down-regulated in TaMAPK4 knockdown plants. Overall, these results suggest that TaMAPK4 plays an important role in signaling during the wheat-Pst interaction. These results present new insights into MAPK signaling in wheat defense to rust pathogen.

Keywords: Puccinia striiformis; host defense mechanisms; mitogen-activated protein kinase; virus-induced gene silencing; wheat.

FIGURE 1

Co-expression of tae-miR164 and TaMAPK4 in Nicotiana benthamiana leaves. (A) β-glucuronidase (GUS) phenotypes observed by histochemical staining. (B) Quantitative detection of GUS activity in leaves inoculated with different recombinant vectors at different time points using a fluorospectrophotometer. EV, empty vector; 4-MU, 4-methyl-umbelliferyl- β-D-glucuronide. The results are presented as the means ± standard errors of three biological replications. Significant differences were determined using Student’s t-test: ^∗P < 0.05.

FIGURE 2

Subcellular localization of TaMAPK4 protein. (A) TaMAPK4-GFP fusion protein and green fluorescent protein (GFP) (control) were transiently expressed in N. benthamiana. (B) Western blot analysis of GFP and TaMAPK4-GFP fusion protein. Bar = 20 μm. Similar results were obtained from three biological replicates.

FIGURE 3

qRT-PCR analysis of relative transcript levels of TaMAPK4. (A) Transcript profiles of TaMAPK4 in wheat leaves (Su11) inoculated with Pst races CYR31 (virulent) and CYR23 (avirulent), respectively. (B) TaMAPK4 transcript profiles in wheat leaves treated with hormone elicitors. SA, salicylic acid; MeJA, methyl jasmonate; ET, ethylene. Mean expression values were calculated from three independent replicates. Significant differences were determined using Student’s t-test: ^∗P < 0.05.

FIGURE 4

Functional characterization of TaMAPK4 after virus-induced gene silencing. (A) Phenotypic changes in the fourth leaves of plants pre-inoculated with positive control vector (BSMV-PDS), FES buffer (CK), or empty BSMV vector (BSMV-γ) at 14 days post-virus treatment. Phenotypes for the fourth leaves inoculated with Puccinia striiformis f. sp. tritici (Pst) races CYR23, or CYR31 at 14 dpi. (B) Relative transcript levels of TaMAPK4 in TaMAPK4 knockdown leaves. RNA samples were isolated from TaMAPK4 knockdown leaves infected with Pst races CYR23 (left), or CYR31 (right). BSMV-γ leaves infected with Pst races CYR23, or CYR31 were used as control, respectively. (C) Biomass of Pst (CYR23) measured at 24 and 120 hpi. Means and standard deviations were calculated from three independent replicates. Significant differences were determined using Student’s t-test: ^∗P < 0.05.

FIGURE 5

Salicylic acid accumulation and transcript levels of pathogenesis-related (PR) genes in TaMAPK4 knockdown leaves. (A) SA accumulation in TaMAPK4 knockdown leaves inoculated with Pst race CYR23. SA, salicylic acid; ng/g, SA accumulation (ng) per fresh leaves weight (g); (B) Transcript levels of TaPR1, TaPR2, and TaPR5 genes in TaMAPK4 knockdown leaves challenged with Pst race CYR23. TaPR1, pathogenesis-related protein 1; TaPR2, β-1,3-glucanase; TaPR5, thaumatin-like protein. BSMV-γ leaves infected with Pst race CYR23 were used as control. Means and standard deviations were calculated from three independent replicates. Significant differences were determined using Student’s t-test: ^∗P < 0.05.