Progress 08/01/24 to 07/31/25
Outputs
Target Audience:The target audience for this project is the mycorrhizal symbiosis research community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student, Elizabeth Trost has been working on characterization and analysis of the Kinase C proteins and also the PT4 phosphosite mutants Through this project, Lizzie has learned how to establish and analyze AM symbioses between M. truncatula and various AM fungi. She has also developed cloning and gene mutation skills, root transformation and confocal microscopy skills. She has become familiar with AM symbioses and their significance for the environment and potential for use in agriculture. The project also provided opportunities for a former postdoc, Sergey Ivanov, to further develop the complex split-YFP system. He had generated the first set of vectors for cytosolic proteins as part of another project (PGRP). Here, he further developed the approach for assessment of membrane protein-protein interactions in colonized root cells. How have the results been disseminated to communities of interest?Elizabeth Trost has presented data on the Kinase C mutants and also the PT4 phosphosite mutants in two talks and two posters. Short talks: E. Trost, PLPPM 6820 Research Updates, The proceedings of the Plant Pathology & Plant-Microbe Biology Graduate Student Seminar, April 18th, 2025. E. Trost, Characterizing the role of the KINASE Cs in arbuscular mycorrhizal symbiosis, The proceedings of the Cornell Fungal Supergroup, August 20, 2024. Posters: E. Trost, KINASE C is essential for arbuscular mycorrhizal symbiosis, The proceedings of the Boyce Thompson Centennial Symposium, August 8-9, 2024. E. Trost, KINASE C is essential for arbuscular mycorrhizal symbiosis, The proceedings of the Cornell SIPS 10-year Anniversary Symposium, October 8, 2024 What do you plan to do during the next reporting period to accomplish the goals?We will continue our experiments to address the objectives as outlined in the original proposal.
Impacts
What was accomplished under these goals?
Under objective 1, we have initiated characterization of proteins predicted to interact with PT4 using Bifluorescence Complementation assays (BIFC). These assays are carried out in M. truncatula mycorrhizal roots in colonized cells where we assess interaction of PT4 in its native location at the periarbuscular membrane with the native level of expression. In order to do this, PT4 -split YFP fusions and candidate protein-split YFP fusions are expressed from their native promoters. The constructs are carried on a single T-DNA which also carries a transformation marker so that we can ensure that the cells are transformed and colonized. The latter is important for interpreting constructs that do not result in a YFP signal. The transformation marker is a nuclear-localized-mCherry driven from a mycorrhiza-inducible promoter, thus transformed, colonized cells have a red fluorescent nucleus. We have completed development of the vector backbone and the first interaction studies. We evaluated the new vector system using STR-STR2 proteins, which we showed many years ago, interact with each other at the periarbuscular membrane. The vector system worked well and we moved on to evaluate M. truncatula HA1, a proton-ATPase. M. truncatula HA1 was one of the most consistent PT4 interactors in the co-immunoprecipitation mass spec analysis. We have confirmed that MtHA1 is induced during AM symbiosis and located on the periarbuscular membrane, then we generated all potential gene fusions ie., N and C terminal fusions with the N and C terminal halves of YFP. These were combined with all possible combinations of PT4 split YFP fusions. The constructs were transformed into M. truncatula and the composite plants inoculated with Rhizophagus irregularis. The first interaction results are very promising; split YFP analyses showed that PT4 can interact with HA1. We see interaction in both directions, with stronger signals are when HA1 is tagged on the N-terminal end and PT4 on the C-terminal end. This is consistent with current knowledge of proton pump regulation, as the proton-pump is postranslationally modified in its C-terminal regions. The PT4-HA1 interaction occurs at the periarbuscular membrane, the native location of both proteins. As PT4 is a proton-coupled symporter, it is possible that the interaction with HA1 is significant for function. This will be assessed and the next experiments will involve analysis of the PT4 mutant proteins with HA1. The generation of these constructs is underway. Under objective 2, we have built on our initial characterization of the kinase C mutants. The mutants were generated by CRISPR-Cas editing and we have two independent alleles of kinc1, two independent alleles of kinc2 and two independent kinc1 kinc2 double mutants. We have completed analyses with two AM fungi, Rhizophagus irregularis and Diversipora epigaea and confirmed our preliminary results presented in the proposal. The single mutants do not differ significantly from wild type, however colonization of the double mutant is drastically impaired, and arbuscules fail to develop fully and die prematurely. Additionally, we extended the analysis to assess colonization under low- and high-nitrogen regimes. These analyses revealed that in nitrogen starvation conditions, the arbuscule degeneration phenotype is suppressed and fully developed arbuscules are observed. Interestingly, this mirrors the pt4 premature arbuscule degeneration phenotype which is also suppressed in low nitrogen conditions. These data further strengthen a potential link between PT4 and the Kinase C proteins. In addition to phenotyping, we have obtained CAS9-free double mutants and these will allow further interrogation of the Kinase C functions through complementation analyses. To further address the roles of the Kinase C proteins, we have generated several mutant versions of the Kinase C proteins to be tested for their ability to complement the double mutant. We have a phosphoproteomics dataset (M. truncatula/Diversispora epigaea mycorrhizal roots, generated through a DOE user facility project) and this revealed a phosphorylated serine in KINC1 and KINC2. The serine is located in the juxta membrane region, a region that has been shown to be important in some other kinases. To evaluate the contribution of this phosphoserine, we generated phosphate ablative (serine to alanine mutation) and phosphomimetic (serine to aspartic acid) mutants for each kinase. The mutated kinases, expressed from their native promoters, were transformed into the kinc1 kinc2 double mutant (Cas9 free) and their ability to complete the mutant phenotype was assessed. In all cases, the mutant proteins complemented the kinc1 kinc2 double mutants so the significance of the phosphoserine remains to be determined. Experiments to assess the impact of the phosphoserine residue in the low nitrogen aspect of the phenotype will be assessed next. In addition to the phosphoserine mutants, we generated active site mutants of each kinase, one of which is predicted to result in loss of kinase activity (kinase dead) and the other is potentially constitutively active. These constructs have been transformed into the M. truncatula kinc1 kinc2 double mutant and the impact of these mutations will be assessed in the next couple of months. If we are able to obtain a constitutive-active version of these kinases, we will also assess the effect of these proteins in the wildtype background, where a dominant gain of function allele will aid in determining the roles of the Kinase C proteins during symbiosis. Our phosphoproteomic dataset also revealed phosphorylated residues in PT4 and we have evaluated the impact of these residues on PT4 function. In a similar manner as described above, phosphoablative and phosphomimetic versions of the PT4 were generated, transformed in the pt4 mutant and the impact on AM symbiosis was evaluated. Interestingly, the PT4 variant with a phosphoablative mutation at serine 506 failed to complement pt4 indicating that this residue, in the C-terminal cytosolic domain, that is important for function. This is an exciting result and we will integrate this mutant into the interaction studies (Obj1) and also evaluate the kinases for their ability to phosphorylate this residue. Under objective 3, we are assessing the function of a fungal phosphate transporter from the AM fungus, Diversispora epigaea, via host-induced-gene silencing. These constructs are in the queue for transformation for the generation of stable transgenic lines.
Publications
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