pGG-PIP: A GreenGate (GG) entry vector collection with Plant Immune system Promoters (PIP)

  1. Jacob Calabria
  2. Madlen I. Rast-Somssich
  3. Liu Wang
  4. Hsiang-Wen Chen
  5. Michelle Watt
  6. Alexander Idnurm
  7. Staffan Persson
  8. Marc Somssich

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Abstract

The regulatory sequences controlling the expression of a gene (i.e., the promoter) are essential to properly understand a gene’s function. From their use in mutant complementation assays, to studying their responsiveness to different stimuli via transcriptional reporter lines or using them as proxy for the activation of certain pathways, assays using promoter sequences are valuable tools for insight into the genetic architecture underlying plant life. The GreenGate (GG) system is a plant-specific variant of the Golden Gate assembly method, a modular cloning system that allows the hierarchical assembly of individual donor DNA fragments into one expression clone via a single reaction step. Here, we present a collection of 75 GG entry vectors carrying putative regulatory sequences for Arabidopsis thaliana genes involved in many different pathways of the plant immune system, designated Plant Immune system Promoters (PIP). This pGG-PIP entry vector set enables the rapid assembly of expression vectors to be used for transcriptional reporters of plant immune system components, mutant complementation assays when coupled with coding sequences, mis-expression experiments for genes of interest, or the targeted use of CRISPR/Cas9 genome editing. We used pGG-PIP vectors to create fluorescent transcriptional reporters in A . thaliana and demonstrated the potential of these reporters to image the responsiveness of specific plant immunity genes to infection and colonization by the fungal pathogen Fusarium oxysporum . Using the PLANT ELICITOR PEPTIDE (PEP) pathway as an example, we show that several components of this pathway are locally activated in response to colonization by the fungus.

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    Reply to the reviewers

    Reviewer #1: Major comments: The key point of the manuscript is to provide resources for the plant community. The motivation for selecting these specific promoters, how they were obtained and cloned, what they are in detail and how they will be made publically available is all clearly described. The infection experiments presented in it are an added bonus and a proof of concept of the applicability of the system.

    Thank you very much.

    Minor comments: The promotor sequences will probably be included in the AddGene submission, however, it might be helpful to also deposit the promoter sequences at e.g. GenBank.

    Indeed, we have sent all sequence files to AddGene and they will be available for download there. We will look into transferring them to GenBank as well. We have not done this before, but are generally always supportive of maintaining data in open repositories.

    Line 133: "There are few exceptions to this rule...". It would probably helpful to list/mark these exceptions in Table 1

    We agree. We have now marked them in the table, and included the sentence “There are a few exceptions to this rule (marked with a * in the ‘Bases’ column in table 2), where we used a defined stretch of DNA that has previously been described to complement a mutant” in lines 135-137.

    Line 138: "A overhangs". In the GreenGate system, A-modules (promoters) are flanked by A- (5') and B- (3') overhangs (applies to line 144, too). Also, the B-overhang listed here (TTGT) is the reverse complement, which might be confusing for readers.

    A very good point. We have modified these lines to “standard four base pair GreenGate promoter module overhangs (5´-ACCT and TTGT-3´) were added via primers during amplification of the promoter sequences (see Supplementary Table 1 for a list of primer sequences. Note that TTGT is the complementary sequence of the A-to-B-module overhang, as this is added via the reverse primer)” in lines 141-144.

    Line 149 ff.: How many lines have been established per promoter tested? Did they all yield a similar expression pattern?

    This is indeed a very important point which was somehow lost along the way during manuscript preparations, after being moved around between results and methods section. We have put it back in in lines 162-165 as “We recovered several independent transgenic lines for the PEP1 and 2, PEPR1 and 2, as well as BIK1 and RBOHD reporters. Out of those, a minimum of three (RBOHD) and up to seven (PEPR2) independent lines showed fluorescence, and out of those, all individual lines for each reporter showed the same expression patterns.”

    Line 163: As someone not being familiar with microscoping Arabidopsis roots, I'm wondering how the authors can be sure that the tissue in question is the vasculature. Is this obvious for experts in the field?

    Of course, we can’t give a totally objective answer here, but we believe that by including the transmitted light image next to the fluorescence image, it is indeed visible that the fluorescence is limited to the center of the root, not the complete circumference. At the same time, it is important to note that all images are stereomicroscopic images, not confocal images. Thus, it is indeed not possible to, e.g., conclude if pericycle cells are included or excluded in the region with expression. So, while it is, we believe, safe to assume that it is vascular cells, we can’t determine which cell types in the vascular cylinder are expressing the reporters. This would require confocal imaging, which would increase the resolution, but at the expense of a good overview, which we think is more valuable for such a proof-of-principle.

    Discussion: Is there by any chance prior (cell-resolution) knowledge about the expression behaviour of any of the investigated promoters? E. g. by in-situ hybridizations? If so, do the expression patterns match?

    No, the expression of these reporters in direct response to fungal infection have so far only been studied by transcriptomics.

    Presentation and quality of the images need be improved. Scale bars are missing in all confocal images. In Figure 3 and 4, the name of genes examined can be labeled on the image, which will make it easier for readers. In addition, key information such as the inoculum and sampling time point after fungal inoculation should be described in the legend or the main text.

    We have added the scale bars and gene names into the images. We agree that the gene names make it easier for the reader. Further, we have added the inoculum and sampling time to the legend.

    More importantly, a "mock" inoculation or "before fungal inoculation" should be performed to reveal the expression changes of the marker genes after fungal inoculation.

    This is information was provided in the text and via the supplemental figures, but I assume we didn’t make it clear that these results and images were indeed specific control/mock experiments, and not some ‘general’ expression analysis. We have now tried to make this clearer, specifically in lines 192-194.

    Lines 172-174, the pictures are too small to see these details. The same for BIK1 (line 187).

    We have split up figure 3 into two separate figures (figures 3 and 4), to allow for them to be displayed larger, so that more details can be observed. Of course, it would also be helpful to do some confocal microscopy on specific regions of interest of these stereomicroscopic images to obtain high-resolution images of these regions, but, unfortunately, we did not reach this point in this project, before our team was disbanded, and we therefore only have the overview images to get a general idea of the responsiveness of the different reporters.

    Line 174-176, which results are these referring to? The same for line 200-203.

    We assume that this was not clear because we previously failed to make it clear that the control supplementary figures are from experimental controls/mock. We have reworded both paragraphs to, hopefully, explain it a bit better, and included the supplementary figure number that refers to. It’s now in lines 212-215 and 237-242.

    This study provides a valuable collection of vectors/constructs for investigation of transcriptional dynamics of plant immunity genes and should attract broad interest of the plant immunity field.

    Thank you very much.

    The current study by Calabria et al., entitled "pGG-PIP: A GreenGate (GG) entry vector collection with Plant Immune system Promoters (PIP)," reported the development of a set of GreenGate-compatible entry plasmids that contain promoter sequences of a series of immunity-related genes. This tool enables live-cell observation of immune responses at a cellular resolution. Being compatible with many other GreenGate tools, it opens up a door toward simultaneous visualization of different but overlapping immune pathways and ultimately describes the 4D dynamics of plant immunity. It is more than expected that these constructs will be used by a wide range of researchers and contribute to the ultimate understanding of plant innate immunity.

    Thank you very much.

    It is exciting that the authors observed the marker expression by a fluorescent stereomicroscope. This allows for non-destructive observation of response over time, keeping the system gnotobiotic. However, it was partly disappointing that the author did not take full advantage of this. It would have been much nicer if the authors observed the infection process over time, such that one could tell when and where the response starts, and whether local and systemic reactions occur simultaneously or instead require local-to-systemic signal transduction. They indeed seem to have done such time-course observation (line 378) however did not provide the results. I am curious to know what the authors could have found from those experiments. It would also be a strong appealing point of this method and is therefore highly encouraged

    We absolutely agree that this temporal data would be valuable and interesting. So far, we always imaged the colonization sites in the root tips from the first day when they become visible, until the day when the entire root was colonized/dying. However, we only recorded the infection sites directly, and did not image the entire plants, and local as well as systemic responses. This is, of course, something that we would have liked to do, and planned to do in the future, but, so far, we have not gotten to that point. We also attempted to use the images of the infection sites that we have recorded over time to obtain information about disease progression, e.g., colonization speed of the fungus, but this data is not (yet) at a point, where we feel confident that we have enough information to draw solid conclusions. So, while we absolutely agree that this kind of whole-plant imaging with both, high spatial and temporal resolution, must be the aim, at this point, unfortunately, we simply are not at that place yet.

    Immune responses are not always induction of expression but sometimes reduction. Some genes up-regulated in the first phase will also be down-regulated afterward in order to go back to the initial non-responding state. During such down-regulation, the expression of a fluorescence marker gene might not accurately reflect the real expression levels, because the translated proteins might stay longer even while its transcription is suppressed. To address this point, it is suggested that the authors observe the marker lines in the presence of a translation inhibitor, such as cycloheximide, and quantitatively analyze the dynamics of protein degradation when no new protein is synthesized.

    This is indeed an excellent point. Unfortunately, we have to first say that due to funding issues we are currently unable to do this experiment. However, we did include two things in the revised manuscript: First, we have put in a note that this is indeed a caveat of the system that must be acknowledged (lines 334-337). Second, we have included some information from a different study, which at least addresses this point to some degree. We have imaged the transcriptional response of the *WRKY11 *transcription factor in response to colonization by Fo5176, and in this case, we not only see a local upregulation next to the colonization site, but we see a complete switch in expression pattern. As part of this switch, WRKY11 expression, which was expressed in all root tissues and cells in uninfected control experiments, switches expression off in all tissues and cells except the vascular cells close to the infection site. So here, we indeed have a downregulation of the reporter. In these experiments, signal from the fluorescent WRKY11 reporter disappears from the cells within a day. As we imaged once per day, we can, unfortunately not get more specific than this one-day window. The day before colonization of the tip, signal is seen in all tissues, one day later, if/when the vasculature if colonized in the tip, there is no weak/residual fluorescence left in the cells of the outer tissues. So we can at least state that we would probably also detect downregulation of expression, despite the protein lifetime. Importantly, all our imaging is done on a regular stereomicroscope, and thus, camera sensitivity is moderate. I could imagine that we may be able to detect some residual fluorescence with ultra-sensitive cameras at a spinning disc, or a sensitive detector at a laser-scanning microscope, but we have not tested this. We have added this information in lines 337-347. I apologize that we can’t add more information than this.

    It is remarkable that the authors managed to clone 75 promoter sequences. However, whether all promoters work as expected was not clearly assessed in the present study. Did the authors only transform plants with PEP1, PEP2, PEPR1, and PEPR2 marker constructs? How would they know that the other promoters also work appropriately? In terms of providing these constructs to the research community, it is needed to disclose to which extent the expression has been validated in planta and which promoter has not been assessed.

    This is indeed important information. We have not used the promoters in mutant complementation assays, and have added this caveat in lines 348-350.

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    Referee #3

    Evidence, reproducibility and clarity

    The current study by Calabria et al., entitled "pGG-PIP: A GreenGate (GG) entry vector collection with Plant Immune system Promoters (PIP)," reported the development of a set of GreenGate-compatible entry plasmids that contain promoter sequences of a series of immunity-related genes. This tool enables live-cell observation of immune responses at a cellular resolution. Being compatible with many other GreenGate tools, it opens up a door toward simultaneous visualization of different but overlapping immune pathways and ultimately describes the 4D dynamics of plant immunity. It is more than expected that these constructs will be used by a wide range of researchers and contribute to the ultimate understanding of plant innate immunity.

    It is exciting that the authors observed the marker expression by a fluorescent stereomicroscope. This allows for non-destructive observation of response over time, keeping the system gnotobiotic. However, it was partly disappointing that the author did not take full advantage of this. It would have been much nicer if the authors observed the infection process over time, such that one could tell when and where the response starts, and whether local and systemic reactions occur simultaneously or instead require local-to-systemic signal transduction. They indeed seem to have done such time-course observation (line 378) however did not provide the results. I am curious to know what the authors could have found from those experiments. It would also be a strong appealing point of this method and is therefore highly encouraged.

    Immune responses are not always induction of expression but sometimes reduction. Some genes up-regulated in the first phase will also be down-regulated afterward in order to go back to the initial non-responding state. During such down-regulation, the expression of a fluorescence marker gene might not accurately reflect the real expression levels, because the translated proteins might stay longer even while its transcription is suppressed. To address this point, it is suggested that the authors observe the marker lines in the presence of a translation inhibitor, such as cycloheximide, and quantitatively analyze the dynamics of protein degradation when no new protein is synthesized.

    It is remarkable that the authors managed to clone 75 promoter sequences. However, whether all promoters work as expected was not clearly assessed in the present study. Did the authors only transform plants with PEP1, PEP2, PEPR1, and PEPR2 marker constructs? How would they know that the other promoters also work appropriately? In terms of providing these constructs to the research community, it is needed to disclose to which extent the expression has been validated in planta and which promoter has not been assessed.

    Referee cross-commenting

    I agree with reviewer #1 that the authors need to disclose how many independent lines were established and assessed for each construct.

    I also agree with reviewer #2 that the figure and data presentation needs to be improved.

    Significance

    Overall, the current study already provides a widely useful set of tools for plant researchers, and some additional work would further increase its strength and value.

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    Referee #2

    Evidence, reproducibility and clarity

    This study provides a useful toolkit of reporter/marker constructs for investigating the gene expression of many immune-associated genes. The authors further used this toolkit to examine the expression pattern of several immune elicitor/receptor/downstream component genes after the inoculation of a fungal vascular pathogen Fusarium oxysporum. The study provides valuable tools for plant immunity study. I have some comments regarding the experiment design and data presentation as shown below.

    Presentation and quality of the images need be improved. Scale bars are missing in all confocal images. In Figure 3 and 4, the name of genes examined can be labeled on the image, which will make it easier for readers. In addition, key information such as the inoculum and sampling time point after fungal inoculation should be described in the legend or the main text. More importantly, a "mock" inoculation or "before fungal inoculation" should be performed to reveal the expression changes of the marker genes after fungal inoculation.

    Lines 172-174, the pictures are too small to see these details. The same for BIK1 (line 187). Line 174-176, which results are these referring to? The same for line 200-203.

    Significance

    This study provides a valuable collection of vectors/constructs for investigation of transcriptional dynamics of plant immunity genes and should attract broad interest of the plant immunity field.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In their manuscript, Calabria et al. primarily present a collection of 75 plant (Arabidopsis thaliana) promoters cloned by them into the GreenGate system. These promoters represent different pathways of the plant immune system. Exemplarily they used this compilation to check the involvement of several components of the PLANT ELICITOR PEPTIDE (PEP)-pathway in the response of A. thaliana roots to infection with Fusarium oxysporum strain Fo5176 via transcriptional reporters.

    Major comments:

    The key point of the manuscript is to provide resources for the plant community. The motivation for selecting these specific promoters, how they were obtained and cloned, what they are in detail and how they will be made publically available is all clearly described. The infection experiments presented in it are an added bonus and a proof of concept of the applicability of the system.

    Minor comments:

    The promotor sequences will probably be included in the AddGene submission, however, it might be helpful to also deposit the promoter sequences at e.g. GenBank.

    Line 133: "There are few exceptions to this rule...". It would probably helpful to list/mark these exceptions in Table 1.

    Line 138: "A overhangs". In the GreenGate system, A-modules (promoters) are flanked by A- (5') and B- (3') overhangs (applies to line 144, too). Also, the B-overhang listed here (TTGT) is the reverse complement, which might be confusing for readers.

    Line 149 ff.: How many lines have been established per promoter tested? Did they all yield a similar expression pattern?

    Line 163: As someone not being familiar with microscoping Arabidopsis roots, I'm wondering how the authors can be sure that the tissue in question is the vasculature. Is this obvious for experts in the field?

    Discussion: Is there by any chance prior (cell-resolution) knowledge about the expression behaviour of any of the investigated promoters? E. g. by in-situ hybridizations? If so, do the expression patterns match?

    Significance

    As stated above, this manuscript primarily describes a technical resource useful for the plant science community.

    It is GreenGate-based and therefore easily compatible with other GreenGate-based resource collections. Its primary focus is in the area of plant immune research.

    The key audience is plant immunologists. However, also researchers requiring e.g. tissue-specific and/or pathogen-inducible expression might find it helpful.

    My own field of expertise is plant transformation and cloning systems, thus I went over the part dealing with the proof-of-principle only as a non-expert.

  9. Version published to 10.1101/2022.12.20.521163v2 on bioRxiv
  10. Version published to 10.1101/2022.12.20.521163v1 on bioRxiv