Progress 09/01/18 to 08/31/22
Outputs
Target Audience:Scientific audiences were reached by peer-reviewed publications. Undergraduate students, including Hispanic students, were reached via research experiences in the project. Changes/Problems:Management of the SARS-CoV-2 epidemic continued to place significant restrictions on access to the laboratory, though standardized protocols introduced by the university, as well as the availability of vaccines have loosened the restrictions somewhat. The original PI, Brett Tyler, retired on November 30, 2021, and supervision of the project was taken tranferred to Nik Grunwald. What opportunities for training and professional development has the project provided?Two undergraduate researchers, both biochemistry majors, continued their participation in the project as third year students. The two students continued learning, good laboratory practices, best practices for recording methods and data, best practices for reproducibility, and basic microbiological methods, especially how to keep cultures uncontaminated. This included how to culture E. coli and P. sojae, how to grow soybean seedlings, how to produce zoospores from P. sojae, and how to conduct virulence assays. The postdoctoral scholar strengthened her professional skills in experimental design, project management, presentation, and manuscript presentation. How have the results been disseminated to communities of interest?Manuscripts were submitted for publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Conventionally-bred genetic resistance remains an effective, consumer-friendly strategy to control many diseases. This project investigates the ability of pathogens to adapt to new disease resistance genes. The project focuses on the root rot pathogen of soybean, called Phytophthora sojae. This pathogen belongs to the oomycete group of organisms. The knowledge developed from this project will enable plant breeders to design rational strategies for combining disease resistance genes. During this reporting period, genomic investigations and measurements of pathogen virulence confirmed that some Phytophthora sojae strains show higher levels of variability than observed in strains routinely used for laboratory research, which were selected for their stable properties. These observations suggest that these pathogens may be more adaptable, and able to overcome control measures, than previously appreciated. SPECIFIC AIMS AIM (1) Confirm the role of the suppressor genes already identified by over-expression of those genes in P. sojae strains lacking Avh180, and by CRISPR-mediated deletion of the unsilenced genes in the suppressor strains 1.1) Major activities completed / experiments conducted In our previous report, we reported that we had isolated five Avh180 knock-in lines, derived from the knockout line 180-del5, and that one of them, 180-del5-T38 had regained virulence. During this report year we focused on the following activities: Measure Avh180 transcript levels in the five Avh180 knock-in lines derived from the knockout line 180-del5, to determine if Avh180 expression was responsible for restoring virulence to 180-del5. Produce additional Avh180 knock-in lines derived from 180-del5 and 180-del8. Conduct transformation experiments to over-express RIG1 and RIG2 in Avh180 knockout lines 180-del5 and 180-del8, to further test whether over-expression these two genes can compensate for lack of Avh180. Additional RIG genes RIG4, RIG5, RIG6 and RIG7 will be tested in this way also, if the experiments with RIG1 and RIG2 are successful. Use a CRISPR/Cas12 system to knockout RIG1 in the wildtype, P6497 and in 180-del15-R1 to test the function of RIG1. Use the system to knockout RIG2 in P6497 and in 180-del7-R1 to confirm the contribution of RIG2 to the virulence of 180-del7-R1 on unwounded leaves. AIM (2) Examine suppressor genes for changes in their DNA sequence, copy number, small RNA complement, chromatin marks, or DNA methylation, in order to identify how they have become unsilenced; Nothing to Report AIM (3) Screen for additional P. sojae suppressor strains that have recovered virulence on soybean by overcoming the deletion of Avh180 or three other essential effector genes (Avh17, Avh23 and Avh238). 3.1) Major activities completed / experiments conducted In our previous report, we described a screen for P. sojae lines that had adapted to overcome soybean resistance genes used by breeders, including Rps1a, Rps1k, and Rps3a. One line able to overcome Rps1k was tentatively identified. This year we focused on measuring the expression of P. sojae effector gene Avr1k in the line that could overcome Rps1k, to determine if it had become silenced or otherwise inactivated
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Pei, Y., Si, J.R., Navet, N., Ji, P.Y., Zhang, X., Qiao, H.J., Xu, R.F., Zhai, Y., Tyler, B.M., Dou, D.L. (2022) Two typical acyl-CoA-binding proteins (ACBPs) are required for the asexual development and pathogenicity of Phytophthora sojae Fungal Genetics and Biology, 161:103695. DOI: 10.1016/j.fgb.2022.103695
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Scientific audiences were reached by a peer-reviewed publication. Undergraduate students, including Hispanic students, were reached via research experiences in the project. Changes/Problems:Management of the SARS-CoV-2 epidemic has continued to place significant restrictions on access to the laboratory, though standardized protocols introduced by the university, as well as the availability of vaccines have loosened the restrictions somewhat. Several technical issues also had to be overcome, slowing progress. 1. Transformation efficiencies of knockout lines were initially quite poor. Suspecting that the silenced G418 resistance gene carried by 180-del4, 180-del5 and 180-del8 was interfering with selection of new transformants, we developed a new selectable marker, oxathiapiprolin resistance conferred by a mutant Oxysterol-binding protein-related protein 1 (ORP1) gene. Initial experiments with a P. capsici ORP1 gene produced slow-growing transformants, but use of the P. sojae ORP1 gene enabled us to produce large numbers of normally growing transformants. 2. The yield of knock-in transformants from knockout lines was very low. Suspecting that the Cas9 gene already carried by the knockout lines might be in a silenced state and that silencing might be extending to the incoming Cas9 gene on the knock-in vector, we increased 5-10fold the amount of Cas9 plasmid DNA used for the transformation. This change was successful and also improved the G418 selection. 3. In April 2021 Sigma discontinued selling the protoplasting enzyme Novozyme 234 that we had used for many years for P. sojae transformation. After several months experimentation, we were able to adapt a new protoplasting enzyme mix previously described for P. infestans transformation, though the results remain less reliable than with Novozyme 234. What opportunities for training and professional development has the project provided?Two undergraduate researchers, Jac Longstreth and Jailene Rodriguez, continued their participation in the project as fourth year students. Jac Longstreth, a biochemistry major, graduated in spring 2021 while Jailene Rodriguez, a microbiology major, graduated in Summer 2021. Two new undergraduates joined the team in April 2021, Coranna Akdemirbey and Carla Villarreal; both were nearing the end of their first year as Biochemistry undergraduates. Ms Longstreth and Ms Rodriguez contributed to all aspects of the project, including substantial responsibility for isolating P. sojae lines able to overcome resistance genes. Ms Akdemirbey and Ms Villarreal began learning all necessary safety protocols (including limiting SARS-CoV-2 transmission), good laboratory practices, best practices for recording methods and data, best practices for reproducibility, and basic microbiological methods, especially how to keep cultures uncontaminated. This included how to culture E. coli and P. sojae, how to grow soybean seedlings, how to produce zoospores from P. sojae, and how to conduct virulence assays. Postdoctoral scholar Natasha Navet strengthened her professional skills in experimental design, project management, presentation, and manuscript presentation, and took training courses in bioinformatics and confocal microscopy. How have the results been disseminated to communities of interest?A manuscript describing high frequency silencing Avr1b knock-in transgenes was submitted and accepted for publication. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will test expression levels of Avh180 in the five knock-in transformants derived from knockout line 180-del5. We also plan to focus on transformation experiments to over-express RIG1 and RIG2 in Avh180 knockout lines 180-del5 and 180-del8, to further test whether these two genes can compensate for lack of Avh180. Additional RIG genes RIG4, RIG5, RIG6 and RIG7 will be tested in this way also, if the experiments with RIG1 and RIG2 are successful. We will also conduct knockouts of RIG1 in P6497 and 180-del15-R1 to test the function of RIG1 and confirm its contribution to the ability of 180-del15-R1 to infect unwounded leaves in the absence of Avh180. We will continue to isolate and characterize P. sojae lines able to overcome soybean resistance genes used by breeders.
Impacts
What was accomplished under these goals?
IMPACT Conventionally-bred genetic resistance remains an effective, consumer-friendly strategy to control many diseases. This project investigates the ability of pathogens to adapt to new disease resistance genes, focusing on the oomycete pathogen of soybean, Phytophthora sojae. The knowledge developed from this project will enable plant breeders to design rational strategies for combining disease resistance genes. During this reporting period, genomic investigations and measurements of pathogen virulence suggest that oomycete pathogens like P. sojae may be more adaptable, and able to overcome control measures, than previously appreciated. SPECIFIC AIMS AIM (1) Confirm the role of the suppressor genes already identified by over-expression of those genes in P. sojae strains lacking Avh180, and by CRISPR-mediated deletion of the unsilenced genes in the suppressor strains 1.1) Major activities completed / experiments conducted During this report year we focused on the following activities: Conduct complementation experiments to re-introduce Avh180 into non-virulent knockout lines, isolated last year, to test if their loss of virulence was due to lack of Avh180. We attempted both ectopic expression of Avh180 and a "knock-in" strategy in which Avh180 was restored to its normal genomic location. Measure the gene expression profiles of the RIG genes identified to date, and screen cultures of revertant lines for previously observed RIG gene over-expression. Determined the function of RIG2 by characterizing RIG2 knockout and over-expression lines in collaboration with Dr Daolong Dou's lab in China. 1.2) Data collected (a) 35 ectopic transformants were obtained containing Avh180, but the Avh180 gene was expressed in only one. That one transformant remained non-virulent on unwounded leaves inoculated with zoospores or with mycelia. Thus the non-virulence of 180-del4 was likely due to some other defect than loss of Avh180. Five knock-in transformants containing Avh180 were isolated from 180-del5, but none were recovered from 180-del4 or 180-del8. Of the five knock-in transformants, one had regained virulence, but four had not. Currently we are testing the expression level of Avh180 in each of the knock-in transformants. (b) qRT-PCR measurements revealed that in the wildtype, P6497, genes RIG1, RIG2, RIG4a, RIG6 and RIG7 were strongly elevated during leaf infection, at levels from 600-fold to 18,000-fold compared to mycelia. The results also revealed that RIG1 was elevated over 1900-fold in mycelia and zoospores in 180-del15-R1 compared to the wildtype, while RIG2 was elevated over 180-fold in mycelia and zoospores in 180-del7-R1 compared to the wildtype. However, none of the RIG genes were significantly elevated in our available cultures of 180-del7-R2 or 180-del15-R2. ( c) Dr Dou's students knocked out RIG2. Two independent knockout lines showed reduction of zoospore production and cyst germination by about 50% while infection of etiolated hypocotyls was reduced by about 75%. Knockout of RIG2 reduced lipid body production in mycelia by around 5-10fold while we observed that lipid body production in 180-del7-R1, which over-expresses RIG2, was around 5-10fold greater than in wildtype. 1.3) Summary statistics and discussion of results a) The observation that one of the five knock-in transformants of 180-del5 had regained virulence is a step forward in testing our original observations that Avh180 is required for infection of unwounded leaves by zoospores. The next step will be to test the expression levels of Avh180 in the five transformants. b) The observation that all of the RIG genes except RIG5 are strongly expressed during infection is consistent with the hypothesis that the genes contribute to infection and that over-expression of them could compensate for loss of Avh180. The observation that RIG1 is greatly over-expressed in 180-del15-R1 and that RIG2 is greatly over-expressed in 180-del7-R1, confirms the transcriptome experiments performed in April 2017. However, the qRT-PCR data from the current cultures of 180-del7-R2 and 180-del15-R2 did not exhibit over-expression of any RIG genes indicating that the current cultures are not reflective of the cultures of180-del7-R2 and 180-del15-R2 in our possession in 2017 and 2018. c) RIG2 is very important in infection, as well as in growth and development, including lipid body production. Lipid bodies are an important energy source for zoospores and during early infection. Therefore, we hypothesize that RIG2 over-expression provides elevated energy supplies that enable P. sojae strains lacking Avh180 to persist in establishing infection of an unwounded leaf longer than strains that do not over-express RIG2. 1.4) Key outcomes or other accomplishments realized. The RIG gene expression assays (b) and RIG2 functional studies (c) provide support for our original observations that Avh180 is required for infection of unwounded leaves, and that lack of Avh180 can be compensated by over-expression of genes such as RIG1 and RIG2. In the case of RIG2, elevated energy reserves during penetration of the leaf surface by P. sojae zoospore germlings was identified as a potential mechanism underlying compensation. More broadly, the results support the concept that a wide diversity of mechanisms may underly the physiological plasticity of oomycete pathogens such as P. sojae. AIM (2) Examine suppressor genes for changes in their DNA sequence, copy number, small RNA complement, chromatin marks, or DNA methylation, in order to identify how they have become unsilenced. Relevant to our experiments in Activity 1(a), some transgenes show poor expression, even when they are introduced into their native genome location by knock-in transformation, raises important questions about how specific genes are tagged for silencing or unsilencing. We completed a study of gene silencing of the effector gene Avr1b-1. In our standard wildtype, P6497, Avr1b-1 is naturally silenced compared to other wildtype strains such as P7063 which expresses Avr1b-1 normally. We showed that in knock-in transformants in which modified forms of Avr1b-1 were re-introduced into the site of Avr1b-1 in P7063, the incoming versions of Avr1b-1 were silenced in a majority of the transformants. This result suggests that the Avr1b-1 locus, and possibly other genes such as the RIG genes, carries genomic or epigenetic features that make it highly susceptible to silencing. A peer-reviewed paper was published describing this work. The paper also described the methods used in the current project for knock-in complementation of gene deletion strains. AIM (3) Screen for additional P. sojae suppressor strains that have recovered virulence on soybean by overcoming the deletion of Avh180 or three other essential effector genes (Avh17, Avh23 and Avh238). Due to challenges in completing the gene deletions required for this project goal, we modified our approach to more directly test the ability of the pathogen to overcome resistance genes that target specific effectors. We used the zoospore-leaf screening assay originally developed to identify revertants of Avh180 deletions to search for P. sojae strains that could overcome soybean resistance genes used by breeders, including Rps1a, Rps1k, and Rps3a. We isolated lines of P6497 that had successfully infected leaves containing the resistance genes. However, the lines failed to infect resistant leaves when retested, except for one line that had stably acquired the ability to infect leaves containing Rps1k. That line is currently being tested for possible silencing of Avr1k. Although the current results are very preliminary, they suggest that it is more difficult P. sojae to adapt to overcome resistance genes used by breeders than it is to overcome the loss of Avh180. They suggest that the phenotypic plasticity of oomycetes such as P. sojae may not be uniform; some phenotypes may be more plastic than others.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Gu, B., Shao, G.D., Gao, W.X., Miao, J.Q., Wang, Q.H., Liu, X.L. and Tyler, B.M. (2021) Transcriptional variability associated with CRISPR-mediated gene replacements at the Phytophthora sojae Avr1b-1 locus . Front. Microb. 12:645331. doi:10.3389/fmicb.2021.645331.
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:Undergraduate students, including Hispanic students, were reached via research experiences in the project. A peer-reviewed paper was published. Changes/Problems:The most significant challenge this year was the arrival of the SARS-CoV-2 epidemic, which motivated severe restrictions on access to the laboratory from March 2020 onwards until the end of the reporting year. This challenge interacted with our challenge of recruiting a suitably qualified postdoctoral fellow to the project during this year. An excellent postdoctoral fellow, Natasha Navet was recruited to the project, but she did not join until January 2020, halfway through the reporting year Thus, for the first half of the year, progress depended on technician Felipe Arredondo who has only 25% FTE assigned to this project, and on the two undergraduates, while during the second half of the year the team was restricted in their work by the SARS-CoV-2 epidemic. One outcome of these challenges has been that the qRT-PCR experiments planned for this year were deferred to next year. Also, connected with the SARS-CoV-2 pandemic, two visiting scientists who had been contributing to Aim 3, returned home in April 2020, because they and their families were concerned about how poorly the pandemic was being managed in the US. What opportunities for training and professional development has the project provided?Two undergraduate researchers, Jac Longstreth and Jailene Rodriguez, continued their participation in the project as third year students. Jac Longstreth continued as a biochemistry major while Jailene Rodriguez switched to a microbiology major as a result of her participation in this project. During this year, the two students extended their experience of necessary safety protocols (including limiting SARS-CoV-2 transmission), good laboratory practices, best practices for recording methods and data, best practices for reproducibility, and basic microbiological methods. In addition, the two students took on more responsibility and independence for projects aimed at genetically purifying P. sojae strains and determining the stability of their virulence phenotypes. They also learned to conduct basic molecular biology experiments including transformation of E. coli, isolation of plasmid DNA, polymerase chain reaction, and gel electrophoresis. How have the results been disseminated to communities of interest?A manuscript describing the role of histone H3 K27 methylation in regulating Avr1b expression was submitted and accepted for publication. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to focus on complementation experiments re-introducing Avh180 into the new knockout lines. If Avh180 is confirmed to restore the virulence of one or more of the lines, we will conduct transformation experiments to over-express RIG1 and RIG2 in those lines. Also, in order to address the question of why the cultures of 180-?7-R1, 180-?7-R2, 180-?15-R1, and 180-?15-R2 failed to show the levels of RIG gene expression observed in our initial transcriptome experiments, we will screen additional vials of these strains from our liquid nitrogen storage using qRT-PCR, in case the cultures tested in our second transcriptome experiment were erroneous in some way. This focus is justified by the need to make the most of the limited laboratory access that the team has as a result of SARS-CoV-2-motivated restrictions, together with the importance of independently reproducing our initial findings regarding RIG1 and RIG2.
Impacts
What was accomplished under these goals?
IMPACT Conventionally-bred genetic resistance remains an effective, consumer-friendly strategy to control many diseases. This project investigates the ability of pathogens to adapt to new disease resistance genes. The project focuses on the root rot pathogen of soybean, called Phytophthora sojae. This pathogen belongs to the oomycete group of organisms. The knowledge developed from this project will enable plant breeders to design rational strategies for combining disease resistance genes. During this reporting period, genomic investigations and measurements of pathogen virulence confirmed that some Phytophthora sojae strains show higher levels of variability than observed in strains routinely used for laboratory research, which were selected for their stable properties. These observations suggest that these pathogens may be more adaptable, and able to overcome control measures, than previously appreciated. SPECIFIC AIMS AIM (1) Confirm the role of the suppressor genes already identified by over-expression of those genes in P. sojae strains lacking Avh180, and by CRISPR-mediated deletion of the unsilenced genes in the suppressor strains 1.1) Major activities completed / experiments conducted In our previous report. we documented that our cultures of Avh180 knockout lines 180-del7 and 180-del15 no longer exhibited the loss of virulence on unwounded leaves that we had earlier observed. After genetic purification, a sub-culture of 180-del15 was obtained that exhibited loss of virulence on unwounded leaves. However, no sub-cultures of 180-del7 could be found that exhibited stable loss of virulence on unwounded leaves. During this report year we focused on the two following activities: Conduct complementation experiments to re-introduce Avh180 into the non-virulent line of 180-del15, to confirm that its loss of virulence was due to lack of Avh180. This was attempted by both ectopic expression of Avh180 and by a "knock-in" strategy in which the mCherry coding region at the Avh180 locus was replaced by the Avh180 coding region Isolate new Avh180 knockout lines, in case 180-del15 proved intractable to complementation or in case its lack of virulence was not due to loss of Avh180. 1.2) Data collected (a) Despite extensive efforts, no transformants could be obtained from 180-del15 in which Avh180 was expressed ectopically or in which Avh180 was restored by knock-in. We suspect that the silenced G418 resistance gene and silenced Cas9 gene carried by 180-del15 may have interfered with efforts of re-transform this line. (b) Three new Avh180 knockout lines were obtained. 180-del4, 180-del5 and 180del8. After extensive testing of those lines, the three lines were confirmed to be non-virulent when zoospores were inoculated onto healthy leaves. Five additional transformants were obtained from the knockout experiments that had failed to lose the Avh180 gene; these five transformants were kept as controls. 1.3) Summary statistics and discussion of results The results support our earlier observations that Avh180 is required for infection of unwounded unifoliate leaves by P. sojae zoospores. However, it remains to be confirmed that the loss of virulence in the three new knockout lines results from the loss of Avh180; complementation experiments will be required to confirm this. 1.4) Key outcomes or other accomplishments realized. The tentative confirmation that Avh180 is required for infection of unwounded unifoliate leaves by P. sojae zoospores provides a first step towards confirming our earlier conclusions that P. sojae has multiple mechanisms for overcoming the loss of an essential effector gene. AIM (2) Examine suppressor genes for changes in their DNA sequence, copy number, small RNA complement, chromatin marks, or DNA methylation, in order to identify how they have become unsilenced; As described in our original grant proposal, this project includes a collaboration with Dr Suomeng Dong of Nanjing Agricultural University. Dr Dong's lab has experience with assays of DNA adenine-N6-methylation, and assays of methylation of histones. 2.1; 2.2) Activities completed and Data collected. In collaboration with Dr Dong, we determined that the histone methylation profile of RIG1 and RIG2 in the wildtype, P6497, were typical of silenced genes, in particular showing high levels of methylation of residue lysine 27 of histone H3 (H3 K27). Furthermore, in a histone H3 K27 methylase (hmt1) mutant of P. sojae, RIG1 showed strongly elevated transcript levels, however RIG2 did not, suggesting that silencing (and hence unsilencing) of RIG1 and RIG2 may involve different mechanisms. The P. sojae effector gene, Avr1b-1, also is silenced in the wildtype strain, P6497, but expressed in genetically near-identical strains such as P6954 and sc10. Our collaborative project showed that, like RIG1, Avr1b exhibits high levels of methylation of histone H3 K27. Furthermore, in a histone H3 K27 methylase (hmt1) mutant of P6497, Avr1b-1 regained the elevated transcript levels observed in strains such as P6954 and sc10. As a result, the hmt1mutation caused P6497 to lose the ability to evade the protection provided to soybean plants by resistance gene Rps1b. 2.3) Summary statistics and discussion of results The results of the experiments with RIG1 and Avr1b-1 revealed that methylation of histone H3 K27 is a key mechanism regulating expression of two genes implicated in pathogenic adaptation by P. sojae. In the case of Avr1b, this effector gene is directly silenced so that the pathogen can evade resistance gene-mediated protection. In the case of RIG1, our preliminary data suggest that unsilencing of RIG1 may enable the pathogen to evade a resistance gene that targets the effector Avh180. 2.4) Key outcomes or other accomplishments realized An epigenetic mechanism, methylation and unmethylation of histone H3 K27, was identified as an important mechanism contributing to pathogenic adaptation in P. sojae, and potentially in other oomycete pathogens. A peer-reviewed paper describing the Avr1b work was published. AIM (3) Screen for additional P. sojae suppressor strains that have recovered virulence on soybean by overcoming the deletion of Avh180 or three other essential effector genes (Avh17, Avh23 and Avh238). 3.1) Major activities completed / experiments conducted During this year efforts were continued to obtain CRISPR-mediated knockouts of the following effector genes in P. sojae: Avh17, Avh238, Avh241, Avh172, and Avr3b. CRISPR/Cas9 plasmid constructs needed for the knockouts were constructed. However, this work was suspended when the two visiting researchers conducting the work returned to their home country following the arrival of SARS-CoV-2 in Oregon. 3.2) Data collected - Nothing to report 3.3) Summary statistics and discussion of results - Nothing to report 3.4) Key outcomes or other accomplishments realized. - Nothing to report
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Wang, L.Y., Chen, H., Li, J.J. Shu, H.D., Zhang, X.X., Wang, Y.C., Tyler, B.M., Dong, S.M. (2020) Effector gene silencing mediated by histone methylation underpins host adaptation in an oomycete plant pathogen Nucleic Acids Res. 48(4), 28 17901799. https://doi.org/10.1093/nar/gkz1160
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:Undergraduate students, including Hispanic students, were reached via research experiences in the project. Changes/Problems:As documented earlier in the report, we had two unexpected results that set back the progress of the project. The first was the results of the new transcriptome experiment did not agree with the original experiment conduced prior to proposal submission, due either to wrong cultures being used, or to changes in the cultures recovered from liquid nitrogen. The second unexpected result was that the cultures of the knockout lines 180-del7 and 180-del15 recovered from liquid nitrogen no longer showed the lack of virulence on healthy leaves we had previously observed. Although we eventually recovered a non-virulent sub-culture of 180-del15, much effort was consumed and the line still needs to be re-validated by complementation. An additional challenge was that we were unable to recruit a suitably qualified postdoctoral fellow to the project during this year. Thus progress has depended on technician Felipe Arredondo who has only 25% FTE assigned to this project, and on two first year undergraduates. What opportunities for training and professional development has the project provided?Two undergraduate researchers, Jac Longstreth and Jailene Rodriguez, were recruited to the project as first year students. Both were biochemistry majors. Our preferred practice is to recruit undergraduate researchers as first or second year students so that they can gain the benefits of full immersion into our research team over a period of three to four years. In our experience, this strategy provides a much more impactful experiential learning experience than a brief internship or a single term or single year. During this year, the two students learned all necessary safety protocols, good laboratory practices, best practices for recording methods and data, best practices for reproducibility, and basic microbiological methods, especially how to keep cultures uncontaminated. This included how to culture E. coli and P. sojae, how to grow soybean seedlings, how to produce zoospores from P. sojae, and how to conduct virulence assays. 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?Since we have had difficulty recovering Avh180 knockout lines 180-del7 and 180-del15 with the phenotypes we originally observed, we plan to prepare new knockout lines lacking Avh180 so that we can independently confirm that Avh180 is required for infection of unwounded leaves and so that we can confirm that the RIG genes we earlier identified can in fact compensate for loss of Avh180. The knockout lines will be prepared by using CRISPR/Cas9 to replace the Avh180 gene with the fluorescent protein gene, mCherry. In order to gain confidence that deletion of Avh180 is responsible for loss of virulence in these new knockout lines, we plan to collect multiple transformants that failed to lose Avh180 and test their virulence; this will provide data on how frequently loss of virulence occurs as an outcome of the transformation procedure. To more rigorously confirm that loss of Avh180 is responsible for the loss of virulence in the new knockout lines, and in the recovered non-virulent line of 180-del15, we plan to conduct complementation tests in which the Avh180 gene is re-introduced either by ectopic transformation or by replacement of the mCherry gene with Avh180 (knock-in strategy). In order to gain confidence that restoration of Avh180 is responsible for recovery of virulence in these knockouts, we plan to collect multiple transformants that failed to gain Avh180 and test their virulence; this will provide data on how frequently recovery of virulence occurs as an outcome of the transformation procedure. In order to address the question of why the cultures of 180-del7-R1, 180-del7-R2, 180-del15-R1, and 180-del15-R2 failed to show the levels of RIG gene expression observed in our initial transcriptome experiments, we will screen additional vials of these strains from our liquid nitrogen storage using qRT-PCR, in case the cultures tested in our second transcriptome experiment were erroneous in some way. We will continue with our efforts to knock out effector genes Avh17, Avh238, Avh241, Avh172, and Avr3b. The goal of these experiments is to discover additional effector genes that are strongly required for virulence, suitable for additional reversion experiments.
Impacts
What was accomplished under these goals?
IMPACT Conventionally-bred genetic resistance remains an effective, consumer-friendly strategy to control many diseases. This project investigates the ability of pathogens to adapt to new disease resistance genes. The project focuses on the root rot pathogen of soybean, called Phytophthora sojae. This pathogen belongs to the oomycete group of organisms. The knowledge developed from this project will enable plant breeders to design rational strategies for combining disease resistance genes. During this reporting period, genomic investigations and measurements of pathogen virulence revealed that some Phytophthora sojae strains may show higher levels of variability than observed in strains routinely used for laboratory research, which were selected for their stable properties. These observations suggest that these pathogens may be more adaptable, and able to overcome control measures, than previously appreciated. SPECIFIC AIMS AIM (1) Confirm the role of the suppressor genes already identified by over-expression of those genes in P. sojae strains lacking Avh180, and by CRISPR-mediated deletion of the unsilenced genes in the suppressor strains 1.1) Major activities completed / experiments conducted Prior to conducting CRISPR-mediated deletion experiments, the transcriptome of all strains identified to date was re-measured in a single experiment to strengthen the data already collected, and to confirm the properties of the strains. Strains included the wild type P6497, the Avh180 knockout lines, 180-del7 and 180-del15, and the revertant strains that had regained virulence, 180-del7-R1, 180-del7-R2, 180-del15-R1, and 180-del15-R2. After recovering all the strains from storage in liquid nitrogen, RNA for the experiments was collected from mycelia. After recovering all the strains from storage in liquid nitrogen, the virulence phenotypes of the same strains were also carefully re-tested, particularly the ability of zoospores to infected unwounded leaves. The strains were also genetically purified by isolation of single zoospore lines. The virulence phenotypes of multiple zoospore lines were compared to determine levels of variations among each set of lines. 1.2) Data collected Transcriptome data were collected on the Illumina HiSeq3000 platform in July 2018. Two lanes each yielding approximately 300 million 100 bp single end reads were collected from the 24 libraries, yielding on average 25 million reads per library. Virulence tests consisted of inoculating unwounded soybean unifoliate leaves with a small droplet containing around 200 zoospores. After three days incubation at 100% humidity, the lesions were photographed and the lesion areas determined using ImageJ software. Each virulence test included 5-6 unifoliate leaves. 1.3) Summary statistics and discussion of results Analysis of the RNA sequencing data revealed results discordant with the initial data collected in May 2017. When all of the data (from May 2017 and July 2018) were compared in a single analysis, all of the RIG genes displayed elevated number of transcripts in the revertant samples as previously observed, but not in any of the new samples. These results suggested either that the incorrect strains were used for the July 2018 experiment, or that the transcriptional patterns of the strains had changed after the strains were recovered from liquid nitrogen. Testing of the virulence phenotypes of the strains revealed that zoospores of 180-del7-R1, 180-del7-R2, 180-del15-R1, and 180-del15-R2 all could infect unwounded leaves, as expected. However, zoospores from the original Avh180 knockout lines, 180-del7 and 180-del15, also proved fully capable of infecting unwounded leaves, in contrast to the results obtained at the outset of the project in 2016 that enabled the isolation of 180-del7-R1, 180-del7-R2, 180-del15-R1, and 180-del15-R2. The results again suggested either that the incorrect strains had been recovered from liquid nitrogen, or that the phenotypes of the strains had changed after the strains were recovered from liquid nitrogen. PCR analysis confirmed that the 180-del7 and 180-del15 lines contained the mCherry gene in place of the Avh180 gene, while the transcriptome data showed that neither strain had been accidentally exchanged with a revertant line. Thus the results suggested that phenotypic changes may have occurred during the process of storing the strains in liquid nitrogen and then recovering them into culture. Isolation of single zoospores (which are mononucleate) was used to obtain purified sub-lineages of 180-del7 and 180-del15. The rationale for this approach was that the cultures may contain a mixture of nuclei with different characteristics, some unable to support virulence on unwounded leaves (like the original strains) and some able to support virulence on unwounded leaves (observed in the recovered cultures). Oomycetes like P. sojae consist of a multinucleate syncytium and so can host a mixture of different nuclear types (a heterokaryon). By isolating cultures from single mononucleate zoospores, we aimed to obtain genetically pure sub-cultures from 180-del7 and 180-del15. Both strains yielded a mix of sub-cultures that were non-virulent, partially virulent, or fully virulent. When each sub-culture was repeatedly tested, inconsistent results were obtained. Based on these observations, we hypothesized that both strains had developed a high level of instability even though genetically purified. We do not know if the presence of a Cas9 gene (present from the original Avh180 knockout) is responsible for the instability of the strains. Eventually, a single zoospore sub-culture of 180-del15 was obtained that was stably non-virulent, but none was found for 180-del7. It remains to be determined if the lack of virulence of this 180-del15 sub-culture truly results from the loss of Avh180 or from some other change that occurred during the screening procedure. 1.4) Key outcomes or other accomplishments realized. The work described here indicates that Phytophthora sojae lines can develop unexpected levels of phenotypic instability with respect to virulence, even if they originate from lines that were previously unstable. While it is well known that cultures of P. sojae and of other plant pathogens can undergo changes in virulence (usually loss of virulence) after extended culturing in vitro, the level of instability observed in 180-del7 and 180-del15 was significantly greater. This phenotypic instability, while of unknown genetic or epigenetic basis, suggests that in the field, the pathogen may have a more extensive ability to adapt to challenges such as control measures or host resistance than previously appreciated. The sample may be true of other oomycete pathogens. AIM (2) Examine suppressor genes for changes in their DNA sequence, copy number, small RNA complement, chromatin marks, or DNA methylation, in order to identify how they have become unsilenced; Nothing to report AIM (3) Screen for additional P. sojae suppressor strains that have recovered virulence on soybean by overcoming the deletion of Avh180 or three other essential effector genes (Avh17, Avh23 and Avh238). 3.1) Major activities completed / experiments conducted During this year efforts to obtain CRISPR-mediated knockouts of the following effector genes in P. sojae were initiated: Avh17, Avh238, Avh241, Avh172, and Avr3b 3.2) Data collected - Nothing to report 3.3) Summary statistics and discussion of results - Nothing to report 3.4) Key outcomes or other accomplishments realized. - Nothing to report
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