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.
