Progress 02/29/16 to 02/24/21
Outputs
Target Audience:- The research community studying the interaction of Pseudomonas syringae and other bacteria with plants. - Scientists that are breeding plants for increased resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Under these studies the training of one graduate student, Incheol Yeo, was accomplished. This student is trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. The student is also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. This student, Incheol Yeo, graduated in March, 2020. How have the results been disseminated to communities of interest?For this reporting period, the results described here for Adi3 phosphorylation substrates have been published in the journal PLoS ONE. The paper was published on June 2, 2020: Yeo I-C and Devarenne TP (2020) Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays. PLoS One. 15:e0234011. What do you plan to do during the next reporting period to accomplish the goals?For the Adi3 substrate studies we are working toward identifying Adi3 phosphorylation of RPB2 in vivo, and we are working on a new approach to find phosphorylation substrates for Adi3. This approach involves enzyme catalyzed proximity labeling. In this approach transient interactions with a target protein can be tagged with a marker such as biotin. The biotin-tagged proteins can then be pulled down and identified by MS. We are in the very early stages of these studies and are making the needed constructs. For the TD2 studies we will work toward further confirming the C-terminal cleavage of TD2 and understanding the role of this process in controlling TD2 during the resistance response to P. syringae.
Impacts
What was accomplished under these goals?
For the 2020 reporting period we have been continuing our studies to identify nuclear phosphorylation substrates of Adi3 using peptide microarray chips, which was last reported on in the 2019 report. This approach should help us understand howAdi3 functions in the nucleus to suppress programmed cells death (PCD). In the 2019 report we had described some of the possible Adi3 phosphorylation substrates identified using this process. One of these proteins, RNA polymerase II 2nd largest subunit (RPB2), which is a member of the RNA polymerase II complex that controls transcription of protein-coding genes to procure mRNA. Here we report on our attempts to identify the Adi3 phosphorylation sites on RPB2. By alignments between the phosphorylated Ser peptides that matched RPB2 and the RPB2 sequence we identified several possible Adi3 phosphorylation sites on RPB2: Thr100, Ser102, Ser507, Thr675, Thr676, and Ser679. Each of these amino acids was mutated to Ala individually and in combinations in the E. coli expressed and purified RPB2 protein, and each was tested for loss of phosphorylation by Adi3 using in vitro kinase assays. The results showed that only the T675A and T676A mutants of RPB2 had a loss of phosphorylation by Adi3, indicating these amino acids are real Adi3 phosphorylation sites on RPB2. These results suggest that one way Adi3 may control PCD is through control of gene expression by regulation of RPB2 activity through phosphorylation. The phosphorylation of RPB2 by Adi3 could be a positive or negative regulator of gene expression for PCD control. Future experiments will be geared toward identifying Adi3 phosphorylation of RPB2 in vivo and analyzing the role of this phosphorylation. Over the past few reports we have pursued a new avenue for deciphering the resistance mechanisms employed by tomato for defense against P. syringae. The deamination of threonine by threonine deaminase 2 (TD2) to form alpha-ketobutyrate has been shown to be the rate limiting step in the production of isoleucine (Ile). This Ile can then be used for conjugation to the defense hormone jasmonic acid to generate the active form of jasmonic acid, JA-Ile, for defense against insect herbivores and necrotrophic pathogens. Since JA-Ile can interfere with the salicylic acid (SA) based defenses needed for resistance to P. syringae, plants inhibit JA-Ile-induced gene expression during P. syringae resistance. We have previously reported that TD2 is post-translationally modified within the first 5 minutes after detection of P. syringae flg22. This leads to an inhibition of TD2 enzyme activity. Presumably, this will reduce the JA-Ile levels to prevent interference with SA mediated defenses against P. syringae. Now we report that the TD2 post-translational modification in response to the flagellating peptide flg22 from P. syringae maybe a proteolytic cleavage at the C-terminus of the TD2 protein. This was discovered by isolating TD2 protein from tomato plants after flg22 treatment followed by LC-MS analysis. This analysis showed a large portion of TD2 proteins that were cleaved at residues Phe589 and/or Leu591. Further analysis of how truncation at these residues affects TD2 activity suggests truncation increases TD2 sensitivity to Ile feedback inhibition, which is a mechanism for controlling TD2 enzyme activity. Taken together, our data suggest the TD2 protein may be cleaved in response to flg22 and this modification compromises TD2 enzyme activity by increasing sensitivity to Ile. This would lead to a decrease in the production of Ile and ultimately a reduction in the levels of Ile-JA to allow for full resistance to P. syringae.
Publications
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:- The research community studying the interaction of Pseudomonas syringae and other bacteria with plants. - Scientists that are breeding plants for increased resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Under these studies the training of one graduate student, Incheol Yeo, was accomplished. This student is trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. The student is also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. This student, Incheol Yeo, graduated in March, 2020. How have the results been disseminated to communities of interest?For this reporting period, the results described here for Adi3 phosphorylation substrates have been published in the journal PLoS ONE. The paper was published on June 2, 2020: Yeo I-C and Devarenne TP (2020) Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays. PLoS One. 15:e0234011. What do you plan to do during the next reporting period to accomplish the goals?For the Adi3 substrate studies we are working toward identifying Adi3 phosphorylation of RPB2 in vivo, and we are working on a new approach to find phosphorylation substrates for Adi3. This approach involves enzyme catalyzed proximity labeling. In this approach transient interactions with a target protein can be tagged with a marker such as biotin. The biotin-tagged proteins can then be pulled down and identified by MS. We are in the very early stages of these studies and are making the needed constructs. For the TD2 studies we will work toward further confirming the C-terminal cleavage of TD2 and understanding the role of this process in controlling TD2 during the resistance response to P. syringae.
Impacts
What was accomplished under these goals?
For the 2020 reporting period we have been continuing our studies to identify nuclear phosphorylation substrates of Adi3 using peptide microarray chips, which was last reported on in the 2019 report. This approach should help us understand how Adi3 functions in the nucleus to suppress programmed cells death (PCD). In the 2019 report we had described some of the possible Adi3 phosphorylation substrates identified using this process. One of these proteins, RNA polymerase II 2nd largest subunit (RPB2), which is a member of the RNA polymerase II complex that controls transcription of protein-coding genes to procure mRNA. Here we report on our attempts to identify the Adi3 phosphorylation sites on RPB2. By alignments between the phosphorylated Ser peptides that matched RPB2 and the RPB2 sequence we identified several possible Adi3 phosphorylation sites on RPB2: Thr100, Ser102, Ser507, Thr675, Thr676, and Ser679. Each of these amino acids was mutated to Ala individually and in combinations in the E. coli expressed and purified RPB2 protein, and each was tested for loss of phosphorylation by Adi3 using in vitro kinase assays. The results showed that only the T675A and T676A mutants of RPB2 had a loss of phosphorylation by Adi3, indicating these amino acids are real Adi3 phosphorylation sites on RPB2. These results suggest that one way Adi3 may control PCD is through control of gene expression by regulation of RPB2 activity through phosphorylation. The phosphorylation of RPB2 by Adi3 could be a positive or negative regulator of gene expression for PCD control. Future experiments will be geared toward identifying Adi3 phosphorylation of RPB2 in vivo and analyzing the role of this phosphorylation. Over the past few reports we have pursued a new avenue for deciphering the resistance mechanisms employed by tomato for defense against P. syringae. The deamination of threonine by threonine deaminase 2 (TD2) to form alpha-ketobutyrate has been shown to be the rate limiting step in the production of isoleucine (Ile). This Ile can then be used for conjugation to the defense hormone jasmonic acid to generate the active form of jasmonic acid, JA-Ile, for defense against insect herbivores and necrotrophic pathogens. Since JA-Ile can interfere with the salicylic acid (SA) based defenses needed for resistance to P. syringae, plants inhibit JA-Ile-induced gene expression during P. syringae resistance. We have previously reported that TD2 is post-translationally modified within the first 5 minutes after detection of P. syringae flg22. This leads to an inhibition of TD2 enzyme activity. Presumably, this will reduce the JA-Ile levels to prevent interference with SA mediated defenses against P. syringae. Now we report that the TD2 post-translational modification in response to the flagellating peptide flg22 from P. syringae maybe a proteolytic cleavage at the C-terminus of the TD2 protein. This was discovered by isolating TD2 protein from tomato plants after flg22 treatment followed by LC-MS analysis. This analysis showed a large portion of TD2 proteins that were cleaved at residues Phe589 and/or Leu591. Further analysis of how truncation at these residues affects TD2 activity suggests truncation increases TD2 sensitivity to Ile feedback inhibition, which is a mechanism for controlling TD2 enzyme activity. Taken together, our data suggest the TD2 protein may be cleaved in response to flg22 and this modification compromises TD2 enzyme activity by increasing sensitivity to Ile. This would lead to a decrease in the production of Ile and ultimately a reduction in the levels of Ile-JA to allow for full resistance to P. syringae.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Yeo I-C and Devarenne TP (2020) Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays. PLoS One. 15:e0234011
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience: The research community studying the interaction of Pseudomonas syringae and other bacteria with plants. Scientists that are generating plants for increased resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Under these studies the training training of one graduate student, Incheol Yeo, was accomplished. This student is trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. The student is also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. How have the results been disseminated to communities of interest?For this reporting period, the results described here for Adi3 phosphorylation substrates and the role of TD2 in pathogen resistance have not been disseminated to the scientific community. Both studies have been presented during past reporting periods as posters at scientific meetings. Currently, a manuscript is in development for describing the identification of Adi3 phosphorylation substrates. A manuscript describing the TD2 studies will be prepared in the near future. What do you plan to do during the next reporting period to accomplish the goals?For the Adi3 substrate studies we will confirm phosphorylation sites and try to determine an in vivo function of the phosphorylations. For the TD2 studies we will analyze if TD2 is regulated by other PAMPS in a manner similar to what we have seen for flg22. This will tell us if TD2 is regulated only in response to flg22 or is it a broader PTI response.
Impacts
What was accomplished under these goals?
We have been continuing our studies to identify nuclear phosphorylation substrates of Adi3 using peptide microarray chips, which was last reported on in 2017. This approach should help us understand how Adi3 functions in the nucleus to suppress PCD. In the 2017 report we had described the process of using peptide microarray chips to identify potential Adi3 phosphorylation substrates and how we reduced the potential substrates to a list of the 11 most promising candidates. Those candidate are: RNA polymerase II (2nd largest subunit; RPB2), RNA polymerase IV (2nd largest subunit; NRPD2), a histone demethylase, transcription elongation factor SPTS, RNA polymerase I specific transcription initiation factor RRN3, zinc finger CCCH domain-containing protein 19 NERD, zinc finger CCCH domain protein Oxidative stress 2 (OX2), transcription initiation factor TFIID subunit 11, 26S proteasome regulatory subunit 4 homolog A, apoptotic chromatin condensation inducer, and Pto-interacting 5 Pti5. Now we have analyzed these candidates for phosphorylation by Adi3. The cDNA for each candidate was cloned, expressed in E. coli, the protein purified, and the protein tested for Adi3 phosphorylation using in vitro kinase assays. The cDNA for five of the candidates was not able to be isolated by RT-PCR and two of the candidates did not express in E. coli. This left four of the candidates for which we were able to purify protein and test for Adi3 phosphorylation: RPB2, NRPD2, TFIID, and Pti5. Of these four, only RPB2 and Pti5 were phosphorylated by Adi3. Using site directed mutagenesis and mass spectrometry analysis we have identified the Adi3 phosphorylation sites on RPB2 as Thr69, Thr70, and Ser73. We are in the process of identifying the phosphorylation sites on Pti5. Over the past few reports we have pursued a new avenue for deciphering the resistance mechanisms employed by tomato for defense against P. syringae. The deamination of threonine by threonine deaminase 2 (TD2) to form alpha-ketobutyrate has been shown to be the rate limiting step in the production of isoleucine (Ile). This Ile can then be used for conjugation to the defense hormone jasmonic acid to generate the active form of jasmonic acid, JA-Ile, for defense against insect herbivores and necrotrophic pathogens. Since JA-Ile can interfere with the salicylic acid (SA) based defenses needed for resistance to P. syringae, plants inhibit JA-Ile-induced gene expression during P. syringae resistance. We have previously reported that TD2 is posttranslationally modified within the first 5 minutes after detection of P. syringae flg22. This leads to an inhibition of TD2 enzyme activity. Presumably, this will reduce the JA-Ile levels to prevent interference with SA mediated defenses against P. syringae. Now we report that we have produced a TD2 knockdown (KD) tomato line using RNA silencing and used this line to assess the contribution of TD2 to resistance against P. syringae. It is known that JA-Ile is required for resistance against necrotrophs such as Botrytis cinerea and JA-Ile can counteract resistance to biotrophs/hemibiotrophs such as P. syringae. If TD2 is functioning in the manner we hypothesize and its contribution to JA-Ile production needs to be inhibited for P. syringae resistance, the TD2 KD line should confer greater resistance to P. syringae. In fact, this is what we have seen. Using bacterial growth assays from WT and TD2 KD tomato plants treated with P. syringae we have seen that the TD2 KD plants have an increase in P. syringae resistance by 1 log unit compared to WT plants. Additionally, since JA-Ile is required for resistance to necrotrophs such as B. cinerea, the TD2 KD plants should produce less JA-Ile and be less resistant to B. cinerea. We treated WT and TD2 KD plants with B. cinerea and measured the necrotic halo produced by the fungus after 3 days of growth. The TD2 KD plants showed a large increase in the halo size compared to WT, indicating the TD2 KD plants are less resistant to B. cinerea. These data confirm our hypothesis about how TD2 is functioning. We have also analyzed SA and JA-Ile levels in WT and TD2 KD lines treated with flg22. Additionally, we have analyzed the expression of SA and JA marker genes in WT and TD2 KD lines treated with flg22. The results show that the SA and JA-Ile levels are not significantly different between WT and TD2 KD lines in response to flg22. However, the gene expression analysis showed that the SA marker genes are upregulated in response to flg22 in the TD2 KD plants, which matches our hypothesis and supports the reason for why the TD2 KD plants are more resistant to P. syringae.
Publications
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience: The research community studying the interaction of Pseudomonas syringae and other bacteria with plants. Scientists that are breeding plants for increased resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Under these studies training training of two graduate students, Incheol Yeo and Dongyin Su. These students are trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. They were also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. ? How have the results been disseminated to communities of interest?These results on TD2 have been presented in a poster at Plant Biology, the 2018 annual meeting of the American Society of Plant Biologists. Also, the studies on SnRK1 were published in the journal Biochimica et Biophysica Acta. What do you plan to do during the next reporting period to accomplish the goals?For the TD2 studies we will measure JA-Ile and SA levels in wild-type andTD2 knockdown plants treated with P. syringae. Also, we will analyze resistance to P. syringae inwild-type andTD2 knockdown plants.
Impacts
What was accomplished under these goals?
Studies to characterize the SnRK1 complex from tomato were finalized and published in Biochimica et Biophysica Acta. In these studies we characterized the two alpha subunits of the SnRK1 complex, SnRK1.1 and SnRK1.2. The SnRK1.2 enzyme shows very low kinase activity as compared to SnRK1.1 and has different substrate preference when phosphorylating the SnRK complex beta subunits. Overall, these studies suggest SnRK1.2 has a different in vivo role compared to SnRK1.1. Additionally, we have pursued a new avenue for deciphering the resistance mechanisms employed by tomato for defense against P. syringae. We have found that the enzyme threonine deaminase 2 (TD2) is posttranslationally modified in response to P. syringae flg22. The deamination of threonine by TD2 to form alpha-ketobutyrate has been shown to be the rate limiting step in the production of isoleucine (Ile). This Ile can then be used for conjugation to the defense hormone jasmonic acid to generate the active form of jasmonic acid, JA-Ile, for defense against insect herbivores and necrotrophic pathogens. Since JA-Ile can interfere with the salicylic acid (SA) based defenses needed for resistance to P. syringae, plants inhibit JA-Ile-induced gene expression during P. syringae resistance. We have found that TD2 is posttranslationaly modified within the first 5 minutes after fl22 detection. This leads to an inhibition of TD2 enzyme activity. Presumably, this will reduce the JA-Ile levels to prevent interference with SA mediated defenses against P. syringae.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Su D, Devarenne TP (2018) In vitro activity characterization of the tomato SnRK1 complex proteins. Biochimica et Biophysica Acta - Proteins & Proteomics 1866:857-864.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Yeo I and Devarenne TP (2018) A role for threonine deaminase in tomato plant defense against bacterial infection. Abstract for Plant Biology 2018, annual meeting of the American Society of Plant Biologists, Montreal, Canada. July, 2018.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience: - The research community studying the interaction of Pseudomonas syringae and other bacteria with plants - Scientists that are breeding plansts for increased resistance to pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Under these studies training training of two graduate students, Incheol Yeo and Dongyin Su. These students are trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. They were also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. ? How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will confirm Adi3 phosphorylation of the identified nuclear phosphorylation substrates. This will be accomplished by using mass spectrometry followed by mutation of the identified phosphorylation sites to non- phosphorylation Alanine to show loss of phosphorylation by Adi3. Once confirmed, the role of the phosphorylation on each candidate will be studied in relation to the predicted function of the phosphorylated protein as well as in the context of resistance to P. syringae. Several of the potential Adi3 phosphorylation substrates encode transcription factors or proteins involved in controlling the transcription process raising the possibility of alterations in gene transcription to control PCD in tomato during the resistance response to P. syringae.? For the TD2 studies we will assess the role of PARrylation/dePARrylation on TD2 enzyme activity, identify the PARP responsible for PARylating TD2 and analyze the role of this PARP in pathogen resistance, and use gene silencing techniques to assess the role of TD2 in basal resistance.
Impacts
What was accomplished under these goals?
Studies to identify the nuclear phosphorylation substrates of Adi3 using phosphoproteomics has been very challenging. Thus, over the past year we took a new approach of using peptide microarray chips. These chips contain 1,536 unique synthetic kinase peptide substrates. The peptides on the chips are phosphorylated by Adi3 using [32P]-ATP and peptides phosphorylated by Adi3 are identified by incorporation of 32P. During this process it took several months to optimize the phosphorylation of the peptide chip as there were many factors such as [32P]-ATP concentration, Adi3 concentration, and incubation time that affected the efficiency of phosphorylation. The results showed that Adi3 efficiently phosphorylated many of the peptides on the chip. Using the sequence of the 63 strongest Adi3 phosphorylated peptides to identify proteins with similar sequences to these peptides. Each peptide sequence was BLASTed against the tomato gene and protein databases and returned 1,068 proteins that contained at least 5 amino acids, including the phosphorylatable Ser, identical to any given peptide phosphorylated by Adi3. The highest match of a peptide sequence to a tomato protein was 10 amino acids, which was found for 5 different peptides. Most peptides matched 6 to 8 amino acids in the identified tomato proteins. This list of proteins was further narrowed down to 413 possible candidate proteins based on predicted nuclear localization or relevance to pathogen resistance. This list has been further narrowed to a top 10 list of proteins to be analyzed for Adi3 phosphorylation based on nuclear function and relevance to pathogen resistance. The list includes proteins with functions in chromatin remodeling, transcription regulation, and RNA polymerase. We are currently analyzing Adi3 phosphorylation of these potential substrates. Additionally, this grant has allowed us to pursue a new avenue for deciphering the resistance mechanisms employed by tomato for defense against P. syringae. We have found that the enzyme threonine deaminase 2 (TD2) is posttranslationally modified in response to P. syringae flg22. The deamination of threonine by TD2 to form alpha-ketobutyrate has been shown to be the rate limiting step in the production of isoleucine (Ile). This Ile can then be used for conjugation to the defense hormone jasmonic acid to generate the active form of jasmonic acid, JA-Ile, for defense against insect herbivores and necrotrophic pathogens. Since JA-Ile can interfere with the salicylic acid (SA) based defenses needed for resistance to P. syringae, plants inhibit JA-Ile-induced gene expression during P. syringae resistance. We have found that in response to flg22 TD2 is posttranslationally PARylated, the addition of poly(ADP-ribose) to the protein, this occurs within the first 5 minutes after flg22 detection and is followed by removal of the PARylation by 24 hours after flg22 detection. Our working hypothesis is that TD2 PARylation inhibits TD2 enzyme activity preventing production of the Ile needed for JA-Ile biosynthesis. Thus, the amount of JA-Ile is reduced to prevent interference with the needed SA defense responses. A role for TD2 in the tomato defense response against P. syringae has not yet been shown and our discovery of a potential involvement of TD in the basal defense response to P. syringae flagellin opens an exciting new area of research.
Publications
|
Progress 02/29/16 to 09/30/16
Outputs
Target Audience:- The research community studying the interaction of Pseudomonas syringae and other bacteria with plants. - Scientists that are breeding plants for increased resistance to pathogens. Changes/Problems:There have been no changes or problems at this time. What opportunities for training and professional development has the project provided? Under these studies training of two graduate students, Incheol Yeo and Dongyin Su, has been accomplished. These students are trained in molecular biology and biochemistry techniques to study plant interactions with pathogens. They were also trained in the writing and the publication process for having a scientific study published in an international scientific journal. Additional training includes reviewing manuscripts from scientific journals, creating poster and oral presentations for scientific meetings, developing networking opportunities, and communicating scientific findings to the public. How have the results been disseminated to communities of interest? Over the past year our studies on cell death control and its role in resistance to P. syringae have been disseminated to the general scientific public through the presentation of three posters at Plant Biology 2016, the annual meeting of the American Society of Plant Biologists, Austin, TX. July, 2016. We have also published one paper in the New Phytologist from a collaboration with Dr. Fangming Xiao at the University of Idaho. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period we will confirm Adi3 phosphorylation of the identified nuclear phosphorylation substrates. This will be accomplished by using mass spectrometry followed by mutation of the identified phosphorylation sites to non-phosphorylation Alanine to show loss of phosphorylation by Adi3. Once confirmed, the role of the phosphorylation on each candidate will be studied in relation to the predicted function of the phosphorylated protein as well as in the context of resistance to P. syringae. Several of the potential Adi3 phosphorylation substrates encode transcription factors or proteins involved in controlling the transcription process raising the possibility of alterations in gene transcription to control PCD in tomato during the resistance response to P. syringae. For the TD2 studies we will assess the role of phosphorylation/dephosphorylation on TD2 enzyme activity, identify the TD2 kinase and analyze its role in pathogen resistance, and use gene silencing techniques to assess the role of TD2 in basal resistance.
Impacts
What was accomplished under these goals?
The major accomplishments involved the identification of potential nuclear protein phosphorylation substrates for Adi3. We faced many challenges using a phosphoproteomics and were not successful. Thus, we used peptide microarrays that have over 1,500 phosphorylatable synthetic peptides on a glass slide. The peptides are phosphorylated with Adi3 using [32P]-ATP and the sequence of the phosphorylated peptides are used to search the tomato proteome by BLAST to identify proteins containing amino acid sequences based on the phosphorylated peptides. The Ser peptide microarray chips contain 1,536 individual peptides. Each peptide contains 13 random; 6 amino acids on either side of a phosphorylatable Ser residue. The peptides are printed on the slide in 3 identical subarrays. Each subarray contains 1,536 peptides. Each subarray contains 16 sections and each section contains 96 peptides printed in triplicate. Thus, 16 sections times 96 peptides give 1,536 peptides in each subarray. In this manner, each peptide is printed on the slide 9 times (3 peptides in each subarray) to allow for reproducible results within a single chip. We used the Ser peptide chip for phosphorylation by a constitutively active form of Adi3. There were many parameters to optimize for the phosphorylation of the Ser peptide chip such as the amount of [32P]-ATP to use, the length of incubation for the kinase reactions, the amount of Adi3 kinase to use, and the temperature at which to incubate the reaction. Finding the optimal conditions took 4 attempts at phosphorylating the Ser peptide chip. The conditions used were much different from our typical in vitro Adi3 kinase assay and required 10 times more Adi3 protein and 100 times more [32P]-ATP. Also required was a high definition phosphorimager scan of the phosphorylated chip. This required using a high sensitivity phosphor screen and a new high sensitivity scanning instrument recently purchased by our department. After the 4 standardization assays were done, we performed a final assay to obtain the best phosphorylated Ser peptide chip we could produce. A good phosphorylated chip should clearly distinguish each phosphorylated peptide triplet so that the identity of the peptide can unambiguously be assigned. Identifying the phosphorylated peptides was carried out by the company that manufactures the chips and 63 peptides phosphorylated by Adi3 were identified. Using the sequence information for these 63 peptides phosphorylated by Adi3 we can make some estimates about what amino acid are needed for Adi3 to recognize a phosphorylation site. The results showed there was preference in the peptide substrates to contain aromatic hydrophobic residues such as Tryptophan and Tyrosine. Additionally, Aspartic Acid and Arginine appear to be favored in the +4 position and Methionine in the -4 position of the phosphorylated peptides. Finally, the sequence of the 63 identified peptides phosphorylated by Adi3 was used to identify tomato proteins with similar sequences to these peptides. Each peptide sequence was BLASTed against the tomato gene and protein databases. The results returned 1,068 proteins that contained at least 5 amino acids, including the phosphorylatable Ser, identical to any given peptide phosphorylated by Adi3. The highest match of a peptide sequence to a tomato protein was 10 amino acids, which was found for 5 different peptides. Most peptides matched 6 to 8 amino acids in the identified tomato proteins. This list of proteins was further narrowed down to 413 proteins as possible candidates for followup studies based on predicted nuclear localization or relevance to pathogen resistance. This list of 413 proteins was further narrowed to a list of the top 10 proteins. The cDNA for each of these candidates were cloned and analyzed for phosphorylation by Adi3. Three of these candidates showed potential phosphorylation by Adi3. In the previous reporting period we have shown that the protein threonine deaminase 2 (TD2) is dephosphorylated in response to P. syringae flagellin during the basal resistance response to P. syringae in tomato. The deamination of threonine by TD2 to form alpha-ketobutyrate has been shown to be the rate limiting step in the production of isoleucine (Ile). This Ile can then be used for conjugation to the defense hormone jasmonic acid (JA) to generate the active form of jasmonic acid (JA-Ile) for defense against insect herbivores, but JA-Ile has negative effects on resistance to biotrophs such as P. syringae. Thus, we predict that dephosphorylation of TD2 in response to P. syringae flagellin inactivates TD2 in order to reduce JA-Ile. A role for TD2 in the tomato defense response against P. syringae has not yet been shown and our discovery of a potential involvement of TD2 in the basal defense response to P. syringae flagellin opens an exciting new area of research. In the current reporting period we were able to confirm that TD2 is dephosphorylated in response to P. syringae flagellin using phosphatase inhibitors and TD2 specific and TD2 phosphospecific antibodies. This analysis showed that TD2 is dephosphorylated in response to P. syringae flagellin within in 5 minutes. This is followed by a slow recovery of phosphorylation and full recovery by 24 hours after flagellin treatment. This suggests that TD2 is quickly deactivated in response to flagellin. We also cloned the TD2 cDNA into E. coli expression vectors, and successfully purified TD2 protein to assess the role of TD2 dephosphorylation in TD2 functional assays.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Miao M, Niu X, Kud J, Du X, Avila J, Devarenne TP, Kuhl J, Liu Y, Xiao F. (2016) The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling. New Phytologist. 211:138-148.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Su D and Devarenne TP (2016) Tomato SnRK1 complex functions in pathogen resistance. Abstract for Plant Biology 2016, annual meeting of the American Society of Plant Biologists, Austin, TX. July, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Yeo I-C and Devarenne TP (2016) Screening for nuclear substrates of the tomato protein kinase Adi3, a cell death suppressor, using a peptide microarray approach. Abstract for Plant Biology 2016, annual meeting of the American Society of Plant Biologists, Austin, TX. July, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Yeo I-C and Devarenne TP (2016) Functional role of threonine deaminase 2 in the PAMP response of tomato during defense against bacterial infection. Abstract for Plant Biology 2016, annual meeting of the American Society of Plant Biologists, Austin, TX. July, 2016.
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