Periodic Reporting for period 1 - SCHENGEN-ROOT ('Filling the gaps' in the Schengen pathway for plant root Casparian strip integrity)
Periodo di rendicontazione: 2020-09-01 al 2022-08-31
The funded project addresses a fundamental question with broad impact: How does a cell ensures specific outputs upon perceiving diverse inputs? The ability to respond to external stimuli is a fundamental characteristic of all living organisms.The execution of a myriad of cellular functions thus relies on these pathways to maintain specificity from signal input to cellular output. However, i diverse signalling pathways often share similar or identical intermediate components. This typifies the “hourglass conundrum”, where a multitude of inputs each need to elicit a distinct output via a limited common intermediates. Plants have a de-centralized organization. To adapt to the changing environment, each plant cell must be able to incorporate signals to determine the outputs of growth, development, and immunity. Therefore, plants should have a more pronounced hourglass problem, making them great organisms to understand the mechanisms whereby undesirable crosstalk is avoided and signalling specificity maintained. The overall objective of the SCHENGEN-ROOT project aims to understand how two distinct receptor/ligand signalling pathways maintain output specificity in a single cell type. Using advanced genetic and cell biology approaches, this project dissects signalling specificity with single cell resolution, revealing how common signalling modules can regulate a variety of outputs.
Main results and their exploitation for the whole duration of the project:
1. Endodermis-expressed FLS2 cannot functionally replace SGN3 to fuse Casparian Strip domains.
To study signalling specificity at a single cell level, we developed a tailored genetic background that allows stimulation of two different pathways in Arabidopsis root endodermis.
We employ various assays to evaluate the consequences of two different pathway stimulations upon respective peptide treatments. We compare in detail diffusion barrier integrity , lignin deposition , ROS production, CASP domain fusion and are able to conclude that with flg22 treatment, FLS2 cannot functionally replace SGN3 in the endodermis.
2. Endodermis-expressed FLS2 and SGN3 activate both common and pathway-specific transcriptional responses upon ligand perception.
We conducted comparative RNAseq analysis to analyse the transcriptional profiles upon Flg22 and CIF2 treatments. We found functional overlaps of the two pathways are reflected: genes involved in lignin biosynthesis show matching induction patterns after CIF2 or Flg22 treatments. The scale of overlap of one developmental and one defence pathway is surprisingly extensive: There is a high number of commonly induced genes; and the top 10% of most highly induced genes are in common. Top enriched GO terms reflect general biotic & abiotic stress responses, and the two pathways show matching patterns. But there are genes and GO terms that remain pathway specific.
3. MYB36 is a hub that controls SGN3-specific transcriptional responses.
Our analyses suggested that the transcription factor MYB36, which is a key regulator of endodermis differentiation, acts as a hub to regulate SGN3-specific responses. When we investigated genes that are downregulated in myb36 mutant compared to Col-0, the majority are specifically responsive to CIF2 in our RNAseq.
4. MYB36 phosphorylation and stability is required for CASP domain fusion downstream of the SGN3 pathway.
MYB36 is predicted to be phosphorylated by MPKs. It’s amino acid sequence contains 3 putative MPK phosphorylation motifs. I generated phosphodead variantMYB36AAA, and phosphomimic variant MYB36DDD. MYB36AAA can complement CASP expression in myb36 mutant but failed to complement CASP1 fusion. Further, this phenotype is resistant to CIF2 treatment, suggesting that CIF2 induced domain fusion via activating SGN pathway require these functional phosphosites of MYB36. We also noticed that tags that enhance MYB36AAA stability can partially complement fusion. In contrast, MYB36DDD could fully complement CASP1 domain fusion in myb36 mutant.
5. MAPKs are important to establish CS integrity in the endodermis downstream of SGN3 pathway.
We confirmed that in FLS2endo, both FLS2 and SGN3 pathways could activate MAPK3 and 6 in the endodermis. We map all 20 MPK expressions in Arabidopsis by generating individual transcriptional fluorescent marker lines. We created an ATLAS of all MAPKs and these would provide cellular precision, benefiting various fields in plant science. We found 12 show expression in the endodermis that could be involved downstream of the SGN3 pathway. Single mpk mutants do not show strong defects in CS function.We used an effector HopAI1 of P. syringae, which specifically inactivates multiple MAPKs to suppress host immunity. Upon Est induction of HopAI1 in Col-0 plants, it leads to a delayed barrier formation. But HopAI1 activation in the endodermis does not seem to affect CASP1 domain fusion.
6. Activation of distinct MKKs in the endodermis produces distinct outputs, matching both common and specific outputs of SGN3 and FLS2 pathway.
I introduce sgn3 the constitutively active MKK1a or MKK2a under an endodermal specific promoter. With EST induction, both MKK1 and 2 can improve domain fusion. Quantification demonstrated that the number of holes in CASP1 are reduced significantly. Induction of MKK4a but not MKK5a is sufficient to produce ectopic lignin in sgn3. MKK4 could thus be the common downstream of FLS2 and SGN3. Strikingly, induction of MKK9a suppresses both CASP and lignin accumulation at the CS, but does not affect ectopic lignification. By introducing MYB36 varients into this background, we have evidence that MKK9a’s suppression is directly correlated with MYB36 phosphorylation status and stability. This supports our model that specific phosphorylation and enhanced stability of MYB36 by the SGN3 pathway is essential for its specific output.
1. Endodermis-expressed FLS2 cannot functionally replace SGN3 to fuse Casparian Strip domains.
To study signalling specificity at a single cell level, we developed a tailored genetic background that allows stimulation of two different pathways in Arabidopsis root endodermis.
We employ various assays to evaluate the consequences of two different pathway stimulations upon respective peptide treatments. We compare in detail diffusion barrier integrity , lignin deposition , ROS production, CASP domain fusion and are able to conclude that with flg22 treatment, FLS2 cannot functionally replace SGN3 in the endodermis.
2. Endodermis-expressed FLS2 and SGN3 activate both common and pathway-specific transcriptional responses upon ligand perception.
We conducted comparative RNAseq analysis to analyse the transcriptional profiles upon Flg22 and CIF2 treatments. We found functional overlaps of the two pathways are reflected: genes involved in lignin biosynthesis show matching induction patterns after CIF2 or Flg22 treatments. The scale of overlap of one developmental and one defence pathway is surprisingly extensive: There is a high number of commonly induced genes; and the top 10% of most highly induced genes are in common. Top enriched GO terms reflect general biotic & abiotic stress responses, and the two pathways show matching patterns. But there are genes and GO terms that remain pathway specific.
3. MYB36 is a hub that controls SGN3-specific transcriptional responses.
Our analyses suggested that the transcription factor MYB36, which is a key regulator of endodermis differentiation, acts as a hub to regulate SGN3-specific responses. When we investigated genes that are downregulated in myb36 mutant compared to Col-0, the majority are specifically responsive to CIF2 in our RNAseq.
4. MYB36 phosphorylation and stability is required for CASP domain fusion downstream of the SGN3 pathway.
MYB36 is predicted to be phosphorylated by MPKs. It’s amino acid sequence contains 3 putative MPK phosphorylation motifs. I generated phosphodead variantMYB36AAA, and phosphomimic variant MYB36DDD. MYB36AAA can complement CASP expression in myb36 mutant but failed to complement CASP1 fusion. Further, this phenotype is resistant to CIF2 treatment, suggesting that CIF2 induced domain fusion via activating SGN pathway require these functional phosphosites of MYB36. We also noticed that tags that enhance MYB36AAA stability can partially complement fusion. In contrast, MYB36DDD could fully complement CASP1 domain fusion in myb36 mutant.
5. MAPKs are important to establish CS integrity in the endodermis downstream of SGN3 pathway.
We confirmed that in FLS2endo, both FLS2 and SGN3 pathways could activate MAPK3 and 6 in the endodermis. We map all 20 MPK expressions in Arabidopsis by generating individual transcriptional fluorescent marker lines. We created an ATLAS of all MAPKs and these would provide cellular precision, benefiting various fields in plant science. We found 12 show expression in the endodermis that could be involved downstream of the SGN3 pathway. Single mpk mutants do not show strong defects in CS function.We used an effector HopAI1 of P. syringae, which specifically inactivates multiple MAPKs to suppress host immunity. Upon Est induction of HopAI1 in Col-0 plants, it leads to a delayed barrier formation. But HopAI1 activation in the endodermis does not seem to affect CASP1 domain fusion.
6. Activation of distinct MKKs in the endodermis produces distinct outputs, matching both common and specific outputs of SGN3 and FLS2 pathway.
I introduce sgn3 the constitutively active MKK1a or MKK2a under an endodermal specific promoter. With EST induction, both MKK1 and 2 can improve domain fusion. Quantification demonstrated that the number of holes in CASP1 are reduced significantly. Induction of MKK4a but not MKK5a is sufficient to produce ectopic lignin in sgn3. MKK4 could thus be the common downstream of FLS2 and SGN3. Strikingly, induction of MKK9a suppresses both CASP and lignin accumulation at the CS, but does not affect ectopic lignification. By introducing MYB36 varients into this background, we have evidence that MKK9a’s suppression is directly correlated with MYB36 phosphorylation status and stability. This supports our model that specific phosphorylation and enhanced stability of MYB36 by the SGN3 pathway is essential for its specific output.
This project significantly improved our understanding of how the SGN3 pathway signals to establish the integrity of Casparian strip in the root endodermis. The main aim of the project focused on the fundamental question of specificity. We clearly demonstrated in our system that there is signalling specificity in a single cell. One pathway cannot replace the functional outputs of another when operating in a single cell type. Nevertheless, we observe functional overlaps of the two seeming distinct pathways. Significant transcriptional overlaps from comparative RNAseq analyses indicate a basic functional conservation of diverse receptor/ligand signalling pathways. We therefore could speculate that plants activate the same major responses for most of their pathways, only with small tweaks to sub-functionalize. Such subtle sub-functionalization allows pathways to fine-tune their outputs to fit their specific purposes. In the case of the SGN pathway, it might have evolved from an ancient defence pathway, which could produce ROS and lignin, but it acquires fine-tuning with the actions of MYB36 and CASP proteins to achieve precision of lignin deposition for specific developmental purposes. The precision is essential for developing a continuous apoplastic barrier in the plant roots for efficient nutrient uptake, which is lacking from the FLS2 immunity pathway. We are currently preparing a MS encompassing all the data and our interpretations. With this and our follow up studies, we hope to make a big step forward in understanding signalling specificity in plants.