Competence for transcellular infection in the root cortex involves a post-replicative, cell-cycle exit decision in Medicago truncatula

Reviewer #1 (Public Review):

Many studies reported findings implying that rhizobial infection is associated with cell cycle re-entry and progression, however, our understanding has been fragmented. This study provides exciting new insights as it represents a comprehensive description of the cell cycle progression during early stages of nodulation using fluorescence markers.

To briefly summarize, the authors first monitor H3.1 / H3.3 replacement to distinguish between replicating (S phase) and non-replicating cells to show that M. truncatula cortex cells along the bacterial infection thread are non-replicating (while neighbors enter the S phase). Nuclear size measurements revealed that these non-replicative cells are in the post-replicative stage (G2) rather than in the pre-replicative G1 phase, which the authors confirm with the Plant Cell Cycle Indicator (PlaCCI) fluorescent marker to track cell cycle progression in more detail. Cortex cells in the trajectory of the infection thread did not accumulate the late G2 marker of the PlaCCI nor the G2/M marker KNOLLE, indicating that these cells indeed remain in G2. Because nuclear size measurements indicated that infected cells are polyploid, the authors used the centromere histone marker CENH3 to determine chromosome number. They find that cortex cells giving rise to the nodule primordium are endomitotic and tetraploid, probably because their cell cycle is halted at centromere separation. Although not a focus of this manuscript, the authors also use their fluorescent tools to track cell cycle progression during arbuscular mycorrhiza symbiosis. They confirm that infected cells transition from a replicating to a non-replicating state (H3.1 to H3.3) with progressing development of the arbuscules. In addition, the CENH3 marker confirms previous findings that cortex cells infected by fungi are endocycling (i.e., DNA synthesis without segregation of replicated parts). This represents an important confirmation of previous findings and contrasts with the situation during nodulation symbiosis, where chromosomes separate after replication.

In my view, the part about NF-YA1 is less strong - although I realize this is a compelling candidate to be a regulator of cell cycle progression, the experimental approaches used to address this question falls a bit short, in particular, compared to the very detailed approaches shown in the rest of the manuscript. The authors show that the transcription factor NF-YA1 regulates cell division in tobacco leaves; however, there is no experimental validation in the experimental system (nodules). All conclusions are based on a heterologous cell division system in tobacco leaves. The authors state that NF-YA1 has a nodule-specific role as a regulator of cell differentiation. I am concerned the tobacco system may not allow for adequate testing of this hypothesis. With the fluorescent tools the authors have at hand (in particular tools to detect G2/M transition, which the authors suggest is regulated by NF-YA1), it would be interesting to test what happens to cell division if NF-YA1 is over-expressed in Medicago roots?

Based on NF-YA1 expression data published previously and their results in tobacco epidermal cells, the authors hypothesize that NF-YA regulates the mitotic entry of nodule primordial cells. Given that much of the manuscript deals with earlier stages of the infection, I wonder if NF-YA1 could also have a role in regulating mitotic entry in cells adjacent to the infection thread?

In general, all microscopy images are of very high quality and support the authors' conclusions. While individually each set of fluorescent markers has its limitations, combined they constitute a powerful tool to track various stages of cell cycle progression in individual root cells during symbiosis. Overall, this is a very strong manuscript that comprehensively elucidates root cell cycle changes during microbial infection.

Reviewer #2 (Public Review):

Cell cycle control during nitrogen-fixing symbiosis is an important topic, but our understanding of the process is poor and lacks resolution, as the nodule is a complex organ with many cell types that undergo profound changes. The authors aim to define the cell cycle state of individual plant cells in the emerging nodule primordium, as a transcellular infection thread passes through the meristem to reach cells deep in the incipient nodule and releases bacteria to form symbiosomes. The authors used a number of cell cycle reporters, such as different Histone 3 variants and cyclins, to follow cell cycle progress in exquisite detail. They showed that the host cells in the path of an infection thread exhibit a cell fate distinct from their immediate neighbors: after entering the S phase similar to their neighbors, these cells exit the cell cycle and enter a special differentiated state. This is likely an important shift that allows the proper passage of the infection thread. Although definitive proof needs more investigation, they showed that a pioneering transcription factor, NF-YA1, likely represses these endoreduplicated cells from completing the cell cycle.

Author Response:

We would like to thank you very much for handling and reviewing our manuscript so carefully and to be so positive about our work. We are indeed grateful about these very concise and constructive reviews as well as about the Editorial Assessment. We basically agree with all reviewers' comments. Besides addressing all formal suggestions, we also decided to do some more experiments.

The main concern, the role of the transcription factor NF-YA1 during rhizobial infections, is indeed an absolut valid one. While the CDEL system has its beauties it certainly has its limitations as well. Thus, we will try to assess the role of NF-YA1 during symbiotic infections in Medicago more specifically. We will place NF-YA1 expression under the control of infection-specific promoters to limit pleiotropic effects of ectopic over-expression and assess rhizobial infections as well as cell cycle patterns in tranformed hairy roots producing the H3.1/H3.3 marker. Infection-inducible promoters will also be used to drive the ectopic expression of CYCD3;1 on the cortical infection thread trajectory to locally increase mitotic cycles, in order to test the functional importance of cell cycle exit on cortical infections.

We hope that we will be able to conclude more firmly on NF-YA1 function prior to locking the version of record and to deliver these experiments in a time frame of about 4-6 months, which is the minimum time we need for cloning the respective constructs, doing all hairy root transformations in sufficient numbers and quantitative microscopy.