Two genotypes, one phenotype? The roles of CBP60b during immunity

Responding to pathogenic threats effectively is crucial to ensure a plant’s survival. As such, plants have evolved intricate defense mechanisms that protect them from different kinds of pathogens. Defense responses can be resource-intensive strategies in which, for instance, entire organs are discarded to hinder the spread of an infection to the rest of the plant. These costly but effective strategies are at the root of what we understand as the trade-offs between defense and growth. From an agricultural and breeding perspective, it is of the utmost urgency for us to shed light on the molecular mechanisms underlying the defense responses and trade-offs with growth of plants (Huot et al., 2014; Smakowska et al., 2016).

Plant immunity is classically regarded as a two-tiered response. Firstly, the plant cell senses molecular signatures of the pathogen (known as PAMPs, Pathogen-Associated Molecular Patterns) through membrane receptors. This recognition triggers a first layer of defense mechanisms known as PAMP-Triggered Immunity (PTI; Boutrot and Zipfel, 2017). Second, the plant cell recognizes effector proteins secreted by the pathogen. Detection of pathogen effectors leads to Effector-Triggered Immunity (ETI), which generally unleashes a local hypersensitive response and can prime distal organs of the plant to respond faster and more strongly to similar threats (Yuan et al., 2021).

At the core of ETI signaling resides a set of nucleocytoplasmic receptors known as Nucleotide-binding Leucine-rich Repeat proteins (NLRs). As our knowledge about ETI expands, it becomes increasingly clear that NLRs carry out a multifaceted surveillance task to detect pathogens. NLRs detect the presence of pathogen effector proteins directly, as well as the modification of plant proteins that are targets of pathogen effectors. In the latter scenario, NLRs act as guardians, while the plant proteins subject to NLR surveillance are the guardees (Monteiro and Nishimura, 2018; van Wersch et al., 2020). Despite intensive research on ETI mechanisms in the last two decades, much is still unknown and in need of detailed molecular investigation.

In this issue of Plant Physiology, Lu-Shen et al. report on their study on a small family of atypical transcription factors in Arabidopsis (Arabidopsis thaliana): the Calmodulin-Binding Protein 60 (CBP60) family. Three of the eight CBP60 members in Arabidopsis have been previously characterized within the context of defense, with two of them acting as positive regulators of defense genes, whereas the third acts as a negative regulator (Bouche et al., 2005; Truman et al., 2013). Aiming to expand our knowledge about this family of defense-related transcription factors, Lu-Shen et al. explored the CBP60 members that remained uncharacterized. Among them, CBP60b was selected based on its high expression levels in multiple tissues and stages of plant development during pathogenically unchallenged conditions.

Reverse genetics revealed that, in unchallenged conditions, cbp60b mutants are dwarf, accumulate hydrogen peroxide and salicylic acid, and express higher levels of several defense-related genes. When cbp60b mutants were inoculated with the bacteria Pseudomonas syringae pv. tomato, they showed a higher degree of resistance to infection than wild-type plants which, together with the previous results, indicated that mutating CBP60b triggers autoimmunity in the plant. Based on this assumption, the simplest scenario would imply CBP60b works as a transcriptional repressor of defense genes. The authors used luciferase-based transactivation among other methods and, unexpectedly, demonstrated CBP60b binds to the promoter of defense-related genes and promotes their transcription. Further, the authors generated CBP60b overexpression lines and, in line with the transcriptional activation result, these plants displayed signs of a constitutively overactive immune response (dwarfism, increased expression of defense genes, and pathogen infection resistance). These results suggested the role of CBP60b as a positive regulator of immunity. However, how can absence and excess of a transcriptional activator lead to the same autoimmunity phenotype (Figure 1A)?

Figure 1.

Summary figure of the role of CBP60b on defense. A, Representation of the seemingly contradictory phenotypes obtained in knock-outs and overexpression lines of CBP60b. B, simplified working model for the signaling of CBP60b during defense. CBP60b, CALMODULIN-BINDING PROTEIN 60b; NLR, Nucleotide-binding Leucine-rich Repeat protein; EDS1, ENHANCED DISEASE SUSCEPTIBILITY1; PAD4, PHYTOALEXIN DEFICIENT4; ETI, effector-triggered immunity. Image by Sergio Galindo-Trigo based on Lu-Shen et al. 2021 results.

Based on the current understanding of ETI, the authors proposed CBP60b may be a guardee of the NLR surveillance system. Under this hypothesis, over-activation of defenses in the cbp60b mutant would result from the activation of the NLR cascade in response to the absence of the CBP60b protein. To explore this hypothesis, Lu-Shen et al. elegantly tested the dependence of the cbp60b autoimmunity on the NLR signaling pathway via detailed genetic crosses. In sum, mutating direct downstream elements of NLR signaling (EDS1 or PAD4; Wiermer et al., 2005) in the cbp60b background restored the wild-type phenotype, supporting the authors’ hypothesis. Moreover, the autoimmunity phenotype of the CBP60b overexpression lines was not lost when EDS1 and/or PAD4 were mutated, indicating the shared autoimmunity phenotype in knock-out and overexpression plants is caused by two different signaling pathways.

The authors eloquently propose (1) CBP60b acts as a positive regulator of defense responses helping the plant mount defenses upon pathogen recognition; (2) most likely, the CBP60b or its direct downstream components may be targeted by pathogenic effectors and thus are subject to NLR surveillance; (3) when CBP60b signaling is compromised by the pathogen, the NLR system triggers the ETI response to contain the infection (Figure 1B).

Moving forward it will be interesting to obtain a mechanistic understanding of the connection between CBP60b and NLR signaling to consider translational approaches toward agricultural or breeding efforts. Nevertheless, the work by Lu-Shen et al. has furthered our knowledge about the CBP60 family and shed light on regulatory mechanisms that other members may be subject to as well. Finally, the elegant genetic work presented by Lu-Shen et al. could serve many molecular biology researchers as inspiration on how to overcome seemingly contradictory results and how to interpret initial results carefully.