Getting defensive: Kinase MPK3 offers a target for breeding Fusarium-resistant barley

Plants have multi-layered defense responses to ward off invading pathogens. The first line of defense is initiated by plant recognition of pathogen-associated molecular patterns (PAMPs), also known as PAMP-triggered immunity, which is mediated by plant pattern recognition receptors (DeFalco and Zipfel, 2021). Pattern recognition receptors are usually transmembrane receptor-like kinases or receptor-like proteins that directly interact with PAMPs. The perception of PAMPs by their receptors triggers multiple early defense responses, including calcium flux, activation of mitogen-activated protein kinases (MAPKs), production of reactive oxygen species (ROS), transcriptional reprogramming, callose deposition, and cell-wall strengthening (Kong et al., 2012; Li et al., 2022; DeFalco and Zipfel, 2021).

In early plant defense responses, MAPK cascades play important roles in transducing signals from upstream sensors/receptors to downstream targets. In Arabidopsis (Arabidopsis thaliana), perception of PAMP signals by their receptors leads to the activation of at least two MAPK cascades. MPK3 and MPK6, which are phosphorylated by common upstream kinases, function together in one of the two MAPK cascades. MPK3 and MPK6 are activated in response to all PAMPs reported to date, and their functions have been characterized in several plant species, such as Arabidopsis, rice (Oryza sativa), tobacco (Nicotiana benthamiana), and tomato (Solanum lycopersicum) (Liu et al., 2007; Mao et al., 2011; Kong et al., 2012).

In this issue of the Plant Physiology, Basheer et al. (2022) investigated the function of MPK3 in barley (Hordeum vulgare) during Fusarium graminearum infection. Using genetic, physiological, and proteomic data, their results showed that HvMPK3 knockout mutants exhibit higher root resistance to F. graminearum, possibly due to lower ROS production and higher suberin deposition in roots of the mutants than in wild-type plants (Basheer et al., 2022), thus, providing a potential molecular breeding target for pathogen-resistant barley.

First, the authors used TALEN technology to create HvMPK3 knockout mutants to investigate the function of barley MPK3 (Takáč et al., 2021). The roots of knockout plants displayed strong resistance 24 h and 48 h after inoculation with F. graminearum spores compared to the wild-type plants. In the wild-type plant, the roots were massively occupied and colonized by F. graminearum hyphae, leading to severe epidermal cell death. However, only sparse hyphae were present on the root surface of the knockout mutants, and the amount of dead epidermal cells was considerably low. Compared to the knockout mutant plants, the infected wild-type plants displayed substantially shorter roots with some reddish-brown malformations developed on their surface 10 days after infection. Taken together, these phenotypical analyses revealed that MPK3 knockout could improve barley resistance toward F. graminearum infection.

Since oxidative burst is a predominant early defense response (Xu et al., 2014), ROS production was quantitatively evaluated in wild-type plants and the MPK3 knockout mutants. Fluorescent staining and spectrophotometric quantification revealed that ROS accumulation was substantially higher in the infected roots of wild-type than in the MPK3 knockout mutants. Importantly, ROS signals were not detected in most wild-type root epidermal cells, and the subsequent Propidium iodide-staining assay showed that these cells were already dead after F. graminearum infection. On the contrary, the epidermal cells of HvMPK3 knockout lines remained alive.

To further explore the resistance mechanisms of HvMPK3 against F. graminearum, the authors quantified differentially expressed proteins by proteomics before and after F. graminearum infections in wild-type and HvMPK3 knockout plants. Proteomic analyses were also conducted between F. graminearum-treated lines and the same lines treated with flg22 (Takáč et al., 2021). Compared to flg22, Fusarium treatments distinctly affected proteins involved in lipid-related pathways and ROS regulation. Most importantly, secretory peroxidase levels increased in HvMPK3 knockout lines, and levels of H₂O₂-decomposing enzymes increased remarkably in HvMPK3 knockout lines but decreased slightly in wild-type plants, indicating that the reduced ROS levels in HvMPK3 knockout lines could be explained by the increased capacity to decompose H₂O₂ during Fusarium infection. Interestingly, proteins involved in suberin formation, such as GDSL esterase/lipase and some putative lipid transfer-like proteins, were more abundant in HvMPK3 knockout lines upon Fusarium infection. The authors then examined suberin contents with a fluorescent dye before and after Fusarium infection. Impressively, HvMPK3 knockout lines showed a stronger fluorescent signal in noninfected roots, indicating greater suberin accumulation in the mutants compared to wild-type. After Fusarium infection, the knockout mutants showed a significantly enhanced fluorescent signal, while the wild-type plants only displayed a slight fluorescent signal increase, suggesting that the resistance of HvMPK3 knockout lines may be due to cell-wall stiffening by suberin accumulation on the cell surface of the mutant roots.

In summary, the TALEN-based HvMPK3 knockout mutants display strong resistance against Fusarium infection, providing an excellent genetic modification target for molecular breeding of Fusarium-resistant barley (Figure 1). In-depth study of the mechanism of how MPK3 regulates suberin accumulation will be crucial for expanding the molecular targets for Fusarium-resistant breeding practices. Dissecting the direct phosphorylation substrate of MPK3 should be an effective strategy to reveal this molecular mechanism. In addition, it would be worth studying the function of MPK6 in barley during Fusarium infection, since MPK3 and MPK6 often function redundantly (Mao et al., 2011).

Figure 1.

HvMPK3 knockout lines show resistance to F. graminearum infection 24 h and 48 h after inoculation. A–H, Growth of GFP-expressing F. graminearum hyphae in wild-type plants and HvMPK3 knockout (KO) mutants 24 h after inoculation. Scale bars: 50 µm (I) Quantitative evaluation of the proportion of dead cells in control and infected root apices among tested barley lines. Data are presented as means ± standard deviation (sd); n = 20 roots/line, 70–90 epidermal cells evaluated in each root, error bars represent sd. Statistical significance according to one-way ANOVA. J–M, Growth of GFP-expressing F. graminearum hyphae in wild-type and HvMPK3 knockout mutants 48 h after inoculation. Scale bars: 100 µm. Modified from Basheer et al. (2022), Figure 1.

Funding

This work was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20220417) to Y.Y.

Conflict of interest statement. None declared.