Author response:
eLife assessment
This valuable study reveals how a rhizobial effector protein cleaves and inhibits a key plant receptor for symbiosis signaling, while the host plant counters by phosphorylating the effector. The molecular evidence for the protein-protein interaction and modification is solid, though biological evidence directly linking effector cleavage to rhizobial infection is incomplete. With additional functional data, this work could have implications for understanding intricate plant-microbe dynamics during mutualistic interactions.
Thank you for this helpful comment. In the revised manuscript version, we will be more prudent with directly linking cleavage of Nod factor receptors by NopT and rhizobial infection.
We plan to modify the Title, the One-Sentence Summary, Abstract, and Discussion regarding this point.
Public Reviews:
Reviewer #1 (Public Review):
Bacterial effectors that interfere with the inner molecular workings of eukaryotic host cells are of great biological significance across disciplines. On the one hand they help us to understand the molecular strategies that bacteria use to manipulate host cells. On the other hand they can be used as research tools to reveal molecular details of the intricate workings of the host machinery that is relevant for the interaction/defence/symbiosis with bacteria. The authors investigate the function and biological impact of a rhizobial effector that interacts with and modifies, and curiously is modified by, legume receptors essential for symbiosis. The molecular analysis revealed a bacterial effector that cleaves a plant symbiosis signaling receptor to inhibit signaling and the host counterplay by phosphorylation via a receptor kinase. These findings have potential implications beyond bacterial interactions with plants.
Thank you for highlighting the broad significance of rhizobial effectors in understanding legume-rhizobium interactions. We fully agree with your assessment and will emphasize these points in the revised Introduction and Discussion sections of our manuscript. Specifically, we will expand our Discussion regarding the potential impact of the NopT interaction with symbiotic receptor kinases on plant immune signaling and regarding the general significance of our work.
Bao and colleagues investigated how rhizobial effector proteins can regulate the legume root nodule symbiosis. A rhizobial effector is described to directly modify symbiosis-related signaling proteins, altering the outcome of the symbiosis. Overall, the paper presents findings that will have a wide appeal beyond its primary field.
Out of 15 identified effectors from Sinorhizobium fredii, they focus on the effector NopT, which exhibits proteolytic activity and may therefore cleave specific target proteins of the host plant. They focus on two Nod factor receptors of the legume Lotus japonicus, NFR1 and NFR5, both of which were previously found to be essential for the perception of rhizobial nod factor, and the induction of symbiotic responses such as bacterial infection thread formation in root hairs and root nodule development (Madsen et al., 2003, Nature; Tirichine et al., 2003; Nature). The authors present evidence for an interaction of NopT with NFR1 and NFR5. The paper aims to characterize the biochemical and functional consequences of these interactions and the phenotype that arises when the effector is mutated.
Thank you for your positive feedback on our manuscript. In the revised Introduction and Discussion sections, we plan to better emphasize the interdisciplinary significance of our work. We will show how the knowledge gained from our study can contribute to a better understanding of microbial interactions with eukaryotic hosts in general, which may have a stimulating effect on future research in various research areas such as pathogenesis and immunity.
To ensure that the readers can easily follow the rationale behind our experiments, we will improve the Results section and provide more detailed explanations of how NopT among 15 examined effectors was selected. Additionally, we will provide more background information on NopT and the roles of NFR1 and NFR5 in symbiotic signaling in the Introduction section. As suggested, we will include the references Madsen et al. (2003) and Tirichine et al. (2003) as well as additional references on rhizobial NopT proteins into our revised manuscript version.
Evidence is presented that in vitro NopT can cleave NFR5 at its juxtamembrane region. NFR5 appears also to be cleaved in vivo. and NFR1 appears to inhibit the proteolytic activity of NopT by phosphorylating NopT. When NFR5 and NFR1 are ectopically over-expressed in leaves of the non-legume Nicotiana benthamiana, they induce cell death (Madsen et al., 2011, Plant Journal). Bao et al., found that this cell death response is inhibited by the coexpression of nopT. Mutation of nopT alters the outcome of rhizobial infection in L. japonicus. These conclusions are well supported by the data.
We appreciate that you recognize the value of our data.
The authors present evidence supporting the interaction of NopT with NFR1 and NFR5. In particular, there is solid support for cleavage of NFR5 by NopT (Figure 3) and the identification of NopT phosphorylation sites that inhibit its proteolytic activity (Figure 4C). Cleavage of NFR5 upon expression in N. benthamiana (Figure 3A) requires appropriate controls (inactive mutant versions) that have been provided, since Agrobacterium as a closely rhizobia-related bacterium, might increase defense related proteolytic activity in the plant host cells.
Thank you for recognizing the use of an inactive NopT variant in Figure 3A. In fact, increased activity of plant proteases induced by Agrobacterium is an important point that should not be neglected. We plan to mention this aspect in our revised Discussion.
In the context of your comments, we are planning to make the following improvements to the manuscript:
(1) We will add a more detailed description of the experimental conditions under which the cleavage of NFR5 by NopT was observed in vitro and in vivo.
(2) We plan to provide more comprehensive data on the phosphorylation of NopT by NFR1, including phosphorylation assays and mass spectrometry results. These additional data support the proposed mechanism by which NFR1 inhibits the proteolytic activity of NopT.
(3) We will expand the Discussion on the cell death response induced by ectopic expression of NFR1 and NFR5 in Nicotiana benthamiana. We will include more details from Madsen et al. (2011) to contextualize our findings with published literature.
We believe these additions and clarifications will enhance the clarity and impact of our findings.
Key results from N. benthamiana appear consistent with data from recombinant protein expression in bacteria. For the analysis in the host legume L. japonicus transgenic hairy roots were included. To demonstrate that the cleavage of NFR5 occurs during the interaction in plant cells the authors build largely on western blots. Regardless of whether Nicotiana leaf cells or Lotus root cells are used as the test platform, the Western blots indicate that only a small proportion of NFR5 is cleaved when co-expressed with nopT, and most of the NFR5 persists in its full-length form (Figures 3A-D). It is not quite clear how the authors explain the loss of NFR5 function (loss of cell death, impact on symbiosis), as a vast excess of the tested target remains intact. It is also not clear why a large proportion of NFR5 is unaffected by the proteolytic activity of NopT. This is particularly interesting in Nicotiana in the absence of Nod factor that could trigger NFR1 kinase activity.
Thank you for your comments regarding the cleavage of NFR5 and its functional implications. In the revised version, we will change our manuscript taking into account the following considerations:
(1) We acknowledge that the Western blots indicate only a small proportion of NFR5 is cleaved when co-expressed with NopT. It is worth noting in this context that the proteins were expressed at high levels which likely do not reflect the natural situation in L. japonicus. Low production of cleaved NFR5 in our Western blots with transformed N. benthamiana or L. japonicus cells thus may simply reflect an experimental effect due to high NFR5 protein synthesis. We suggest that the presence of high amounts of intact NFR5 does not have a significant functional impact on plant responses (cell death in N. benthamiana, rhizobial infection of L. japonicus) whereas NFR5 cleavage (or formation of NFR5 cleavage products) may be crucial for the observation of the observed phenotypic changes. The fraction of cleaved NFR5, although small, may be sufficient to disrupt crucial signaling pathways, leading to observable phenotypic changes. We will address possible differences between experimental and natural protein levels in our revised Discussion.
(2) We studied in our work three biochemical aspects of NopT: (i) physical binding of NopT to NFR1 and NFR5 (ii) proteolytical cleavage of NFR5 by NopT and (iii) phosphorylation of NopT by NFR1. These three biochemical properties appear to influence each other. Phosphorylation of NopT by NFR1 appears to reduce its proteolytic activity, thereby counteracting NFR5 degradation by NopT (NFR5 homeostasis). Moreover, as NopT is a phosphorylation substrate for NFR1, NopT probably interferes with kinase mediated downstream responses of NFR1. Thus, NFR5 cleavage activity of NopT appears to be only one feature of NopT. We plan to mention these considerations in our revised Discussion.
It is also difficult to evaluate how the ratios of cleaved and full-length protein change when different versions of NopT are present without a quantification of band strengths normalized to loading controls (Figure 3C, 3D, 3F). The same is true for the blots supporting NFR1 phosphorylation of NopT (Figure 4A).
Thank you for pointing out this aspect. Following your recommendation, we will quantify the band intensities for cleaved and full-length NFR5 in the experiments with different versions of NopT. These values will be normalized to loading controls. Similarly, the Western blots supporting NFR1 phosphorylation of NopT will be quantified. The data for normalized band intensities will be included into the revised figures. The quantifications will provide a clearer understanding of how the ratios of cleaved to full-length proteins change with different NopT variants and also will provide information to which extent NopT is phosphorylated by NFR1.
It is clear that mutation of nopT results in a quantitative infection phenotype. Nodule primordia and infection threads are still formed when L. japonicus plants are inoculated with ∆nopT mutant bacteria, but it is not clear if these primordia are infected or develop into fully functional nodules (Figure 5). A quantification of the ratio of infected and non-infected nodules and primordia would reveal whether NopT is only active at the transition from infection focus to thread or perhaps also later in the bacterial infection process of the developing root nodule.
Thank you for pointing this out. In the revised version of our manuscript, we will provide data showing that there are no obvious differences in nodule formation in plants inoculated with ∆nopT and wild-type NGR234, respectively. However, quantification of infection threads containing our GFP-labeled rhizobia in primordia and nodules would be difficult to perform due to strong autofluorescence signals in these tissues. The main goal of our study was to identify and characterize the interaction between NopT and Nod factor receptors. We therefore believe that an in-depth analysis of the bacterial infection process at later symbiotic stages is out of the scope of the present work.
Reviewer #2 (Public Review):
Summary:
This manuscript presents data demonstrating NopT's interaction with Nod Factor Receptors NFR1 and NFR5 and its impact on cell death inhibition and rhizobial infection. The identification of a truncated NopT variant in certain Sinorhizobium species adds an interesting dimension to the study. These data try to bridge the gaps between classical Nod-factor-dependent nodulation and T3SS NopT effector-dependent nodulation in legume-rhizobium symbiosis. Overall, the research provides interesting insights into the molecular mechanisms underlying symbiotic interactions between rhizobia and legumes.
Strengths:
The manuscript nicely demonstrates NopT's proteolytic cleavage of NFR5, regulated by NFR1 phosphorylation, promoting rhizobial infection in L. japonicus. Intriguingly, authors also identify a truncated NopT variant in certain Sinorhizobium species, maintaining NFR5 cleavage but lacking NFR1 interaction. These findings bridge the T3SS effector with the classical Nod-factor-dependent nodulation pathway, offering novel insights into symbiotic interactions.
We appreciate that you recognize the value of our manuscript.
Weaknesses:
(1) In the previous study, when transiently expressed NopT alone in Nicotiana tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. However, this phenotype was not observed when expressing the same NopT in Nicotiana benthamiana (Figure 1A). Conversely, cell death and a hypersensitive reaction were observed in Figure S8. This raises questions about the suitability of the exogenous expression system for studying NopT proteolysis specificity.
We appreciate your attention to these plant-specific differences. In view of your comments, we plan to revise the Discussion and explain the different expression systems used for studying NopT effects in planta. Previous studies showed that NopT expressed in tobacco (N. tabacum) or in specific Arabidopsis thaliana ecotypes (with PBS1/RPS5 genes) causes rapid cell death (Dai et al. 2008; Khan et al. 2022). Our data shown in Fig. S8 confirm these findings. As cell death (effector triggered immunity) is usually associated with induction of protease activities, we considered N. tabacum and A. thaliana plants as not suitable for testing NFR5 cleavage by NopT. In fact, no NopT/NFR5 experiments were performed with these plants in our study. In contrast, the expression of NopT in Nicotiana benthamiana did not lead to cell death in our experiments. Khan et al. 2022 also reported that cell death does not occur in N. benthamiana unless the cells were transformed with PBS1/RPS5 constructs. Thus, N. benthamiana is a suitable expression system to analyze NopT protease activity on co-expressed substrates. Our revision aims to better understand the advantages of the N. benthamiana expression system for studying NopT mediated proteolysis of NFR5.
(2) NFR5 Loss-of-function mutants do not produce nodules in the presence of rhizobia in lotus roots, and overexpression of NFR1 and NFR5 produces spontaneous nodules. In this regard, if the direct proteolysis target of NopT is NFR5, one could expect the NGR234's infection will not be very successful because of the Native NopT's specific proteolysis function of NFR5 and NFR1. Conversely, in Figure 5, authors observed the different results.
Our inoculation experiments clearly show that NopT of NGR234 has a negative effect on formation of infection foci (Fig. 5A) and nodule primordia (Fig. 5E). Our biochemical analysis indicates that NopT targets the NFR1/NFR5 complex, which most likely impairs activation of downstream responses such as NIN gene expression. Accordingly, NIN promoter activity was found to be higher in roots inoculated with the ΔnopT mutant as compared to the NGR234 wild-type (Fig. 5B and 5D). It is therefore plausible that NopT impairs rhizobial infection of L. japonicus due to inhibition of NFR1/NFR5 functions. We agree with this Reviewer that it can be expected that “NGR234's infection will not be very successful”. Fig. 5 confirms that ΔnopT mutant is indeed a better symbiont and we do not think that we obtained “unexpectedly different results”. In the revised version, we will try to formulate our discussion text better in order to avoid any misunderstandings. Furthermore, will write as figure title “NopT dampens rhizobial infection…” instead of “NopT regulates rhizobial infection…”. We are also considering changing the title of our manuscript.
(3) In Figure 6E, the model illustrates how NopT digests NFR5 to regulate rhizobia infection. However, it raises the question of whether it is reasonable for NGR234 to produce an effector that restricts its own colonization in host plants.
We acknowledge the potential paradox of NGR234 producing an effector that appears to restrict its own colonization in host plants. In fact, depending on the host plant, most rhizobial effectors are “double-edged swords” that play either a positive or negative role in the symbiosis. In response to your comment, we will discuss the possibility that NopT may confer selective advantages in interactions between NGR234 and host plants where NopT plays a positive symbiotic role (Dai et al. 2008; Kambara et al. 2009). Inhibition of NFR1/NFR5 functions by NopT in these host plants could be a feedback response in cells in which symbiotic signaling has already started. It is tempting speculate that the interaction between NopT and Nod factor receptors reduces Nod factor perception and downstream signaling to avoid a possible overreaction of symbiotic signaling, which may result in hypernodulation or formation of empty nodules without bacteria. Furthermore, it is tempting to speculate that NopT targets not only Nod factor receptors but also other host proteins to promote symbiosis, e.g. by suppressing excessive immune responses triggered by hyperinfection of rhizobia. In our revised manuscript, we will highlight the need for further investigations to elucidate the precise mechanisms underlying the observed infection phenotype and the role of NopT in modulating symbiotic signaling pathways.
(4) The failure to generate stable transgenic plants expressing NopT in Lotus japonicus is surprising, considering the manuscript's claim that NopT specifically proteolyzes NFR5, a major player in the response to nodule symbiosis, without being essential for plant development.
Thank you for your comments. The failure to obtain L. japonicus plants constitutively expressing NopT was indeed surprising and suggests that NopT targets not only NFR5 but also other proteins in L. japonicus. The number of NopT substrates in plants could be greater than assumed. For example, we show in our work that NopT can cleave AtLYK5 and LjLYS11. In our manuscript, we don’t provide protocols and data on our efforts to construct L. japonicus plants stably expressing NopT. Indeed, it cannot be completely ruled out that the observed failure is not due to NopT expression, but rather to other factors that influence the transformation and regeneration of explants into whole plants. Our results should therefore not be over-interpreted. We consider a discussion of our failed transformation experiments to be somewhat preliminary and not central to this manuscript. herefore, we plan to modify our Discussion and delete the sentence reporting that stable transgenic plants expressing NopT have not been successfully generated.
