Need help? Recently identified phosphorylation targets of MAP kinase 4 aid plant immunity

Facing the challenge of potential pathogens, plants have evolved a multilayered innate immune system to protect themselves (Chen et al., 2020). The first response of the immune system is initiated by the recognition of conserved pathogen-associated molecular patterns (PAMPs) on the surface of invaded cells, thereby activating PAMP-triggered immunity (PTI; Ngou et al., 2022). Upon PTI induction, PAMPs rapidly induce cytoplasmic Ca²⁺ influx, production of reactive oxygen species, and activation of mitogen-activated protein kinases (MAPKs; Chen et al., 2021). MAPK cascades have been shown to regulate defense gene expression and defense responses in Arabidopsis (Arabidopsis thaliana; Asai et al., 2002; Kong et al., 2012). MAP kinase 4 plays a negative role in salicylic acid (SA)-induced systemic-acquired resistance (SAR) and PTI responses (Petersen et al., 2000).

The CCCH-type zinc finger proteins belong to a superfamily containing tandem zinc-binding motifs involved in many aspects of plant growth, development, and defense response. Recent studies have found that Arabidopsis CCCH paralogous proteins C3H14 and C3H15 are involved in responses to fungal infection (Wang et al., 2020). However, the function of C3H14 and C3H15 in the SA-mediated plant defense response is poorly understood.

In this issue of Plant Physiology, Wang et al. (2022) provide insight into the function of C3H14 and C3H15 in the MPK4-mediated defense pathway. They found that c3h14 c3h15 double mutants exhibit enhanced resistance against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), whereas the single mutants show no difference compared with wild-type plants. In contrast, the transgenic plants overexpressing C3H14 and C3H15 (C3H14OE and C3H15OE) showed increased susceptibility to Pst DC3000. Similarly, the authors found that SAR is compromised in C3H14OE and C3H15OE but enhanced in c3h14 c3h15. In accordance with disease resistance, Pst DC3000-induced SA accumulation and Pathogenesis-Related (PR) gene expression increased in c3h14 c3h15 but decreased in C3H14OE and C3H15OE plants. These results indicate that C3H14 and C3H15 play redundant and negative roles in plant defense against Pst DC3000 and SAR.

NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), also known as NON-INDUCIBLE IMMUNITY 1 (NIM1), functions as a master regulator of SA-mediated plant defense (Figure 1; Chen et al., 2021). NIM-INTERACTING 1 (NIMIN1) and NIMIN2 are two key negative regulators of SA-dependent SAR (Figure 1; Hermann et al., 2013). The authors found that the expression of NIMIN1 and NIMIN2 is reduced in c3h14 c3h15 but increased in C3H14OE and C3H15OE plants compared with wild type (WT), indicating that C3H14 and C3H15 positively regulate NIMIN1 and NIMIN2 expression. C3H14 and C3H15 have a DNA-binding ability and recognize G-rich elements. These findings prompted the authors to test whether C3H14 or C3H15 binds to NIMIN1 and NIMIN2 promoters. Electrophoretic mobility shift assay and chromatin immunoprecipitation quantitative polymerase chain reaction assay confirmed that C3H14 directly binds to NIMIN1 and NIMIN2 promoters. Moreover, the authors found that Pst DC3000 infection inhibited C3H14 binding to NIMIN1 and NIMIN2 promoters. To know whether NIMIN1 and NIMIN2 are required for C3H14-mediated suppression of plant immunity, the authors generated C3H14OE plants in nimin1-1 and nimin2-1 backgrounds. NIMIN1 and NIMIN2 mutation enhanced disease resistance and PR1 gene expression in C3H14OE plants. These findings suggested that NIMIN1 and NIMIN2 are required for the negative role of C3H14 in the immune response to Pst DC3000 infection.

To identify C3H14-interacting proteins, a yeast two-hybrid (Y2H) screening assay was performed by the authors. To this end, MPK4 was found among the C3H14 interactors. Y2H assay further found that C3H14 interacts with MPK4 but not MPK3 or MPK6. In vitro pull-down, in vivo bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed the interaction between C3H14 and MPK4. Since C3H14 interacts with MPK4, it is reasonable to postulate that MPK4 can phosphorylate C3H14. Indeed, flg22 (a 22-amino-acid peptide derived from flagellin) induced the phosphorylation of C3H14 in C3H14OE plants, but this was abolished in C3H14OE mpk4-3 plants.

How does MPK4 influence C3H14-mediated plant defense response? To answer this, the authors tested the Pst DC3000 resistance and defense gene expression in WT, mpk4-3, C3H14OE, and C3H14OE mpk4-3 plants. MPK4 mutation enhanced disease resistance, increased PR1 gene expression, and decreased NIMIN1/2 expression in C3H14OE plants. These results suggested that MPK4 is required for the inhibition of defense response to Pst DC3000 by C3H14. To evaluate how elevated levels of MPK4 affect C3H14-mediated defense response, the authors generated C3H14OE CA (constitutively active)-MPK4 plants (Berriri et al., 2012). They found that the overexpression of CA-MPK4 also enhanced disease resistance to Pst DC3000 in C3H14OE plants. In accordance with this finding, the expression of PR1 was higher and expression of NIMIN1 and NIMIN2 was lower in C3H14OE CA-MPK4 than that in C3H14OE plants. C3H14 was constitutively phosphorylated with or without flg22 treatment in C3H14OE CA-MPK4 plants. Together, these results suggested that MPK4 phosphorylates C3H14, attenuating C3H14-mediated inhibition of defense response to Pst DC3000 and C3H14-activated NIMIN1 and NIMIN2 expression.

In summary, the authors identified CCCH proteins C3H14 and C3H15 as negative regulators of plant immunity against Pst DC3000 (Figure 1). C3H14 binds to the promoters of NIMIN1 and NIMIN2, which are required for C3H14-mediated suppression of plant immunity (Figure 1). The functions of C3H14 and C3H15 in PTI and SAR also depended on MPK4-mediated phosphorylation. Phosphorylation of C3H14 attenuated C3H14-mediated inhibition of defense response to Pst DC3000 and C3H14-activated expression of NIMIN1 and NIMIN2. Although the authors found that MPK4 interacts with C3H14 and mediates its phosphorylation, the phosphorylation site(s) has not been identified yet. In future studies, it will be imperative to identify the phosphorylation site(s) to address the biological function of phosphorylated C3H14 further. Overall, the work by Wang et al. (2022) provides insight into the function of MPK4–C3H14–NIMIN1/2 cascades in plant immunity. The defense mechanism presented by Wang et al. (2022) increases our understanding of the MAPK cascade in plant immunity.

Figure 1.

Schematic model for MPK4–C3H14–NIMIN1/2 cascade in plant defense response. In the absence of pathogen infection, MPK4 is not active and thus cannot phosphorylate downstream signals, and the CCCH-type zinc finger protein C3H14 represses the expression of PAMP-triggered immunity marker genes and activates other target genes to facilitate plant growth (left). The defense response, triggered by Pst DC3000 infection or flg22 treatment, activates MPK4 and promotes the interaction between MPK4 and C3H14, resulting in the phosphorylation of C3H14. The phosphorylation of C3H14 inhibits C3H14-mediated repression of the PTI gene and activation of NIMIN1/2 expression, thereby inhibiting its negative roles in PTI and SAR. The activation of NIMIN1/2 by C3H14 in salicylic acid signaling is independent of MPK4 phosphorylation. The figure was adapted from Wang et al. (2022) and created with BioRender.com.

Funding

This work was supported by grants from Initial Research Fund of Highly Specialized Personnel from Jiangsu University (20JDG34), Natural Science Foundation of Jiangsu Province (BK20211319), and National Natural Science Foundation of China (32000201).

Conflict of interest statement. The author declare no conflict of interest.