Progress 09/01/20 to 08/31/23

Outputs
Target Audience:We published an academic paper describing some of our work on Xanthomonas resistance. This paper will primarily reach an academic audience but is also targetted toward those in industry looking for biotech solutions to control bacterial pathogens in tomato Changes/Problems:The two major problems were difficulty in getting a sufficient number of transformants (we obtained only 125 putative independent events out of a desired 400) and insufficient expression of ZAR1 and JIM2 from the native promoters (two plants containing the desired ZAR1+JIM2 clean insertion event did not expressed the genes to a sufficient level to confer resistance to Xanthomonas). The transformation problem can be overcome in future work by better optimizing the transformation method for these tomato varieties or by using a variety that is more amenable to transformation and then introgressing the insertion into the desired background. Alternatively, a system that can more efficiently generate clean insertions could be used. The expression problem could be overcome by using non-native promoters that give stronger expression. The native promoters were selected because they work in Solanum pennellii, they have fewer regulatory and consumer acceptance concerns than using a strong viral promoter such as 35S. Additionally, ZAR1+JIM2 can give auto activity when highly expressed so we did not want promoters that give expression which is too strong. From Phase I we knew that the native promoters can work, which we also observed here, but unfortunately they only give moderate or strong resistance in about 50% of the transformants (and this could be skewed a by having multiple T-DNA insertions in a plant). Additionally, whereas making traditional transgenics with the desired gene of interest next to the selectable marker tends to select for insertion locations which favor expression of both the gene of interest and the selectable marker, our approach does not have this constraint. We are therefore able to recover plants that have the NPTII selectable marker inserted at a position that is favorable for gene expression whereas the gene of interest may be inserted at a different location that is not conducive for gene expression. What opportunities for training and professional development has the project provided? Nothing Reported 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? Nothing Reported

Impacts
What was accomplished under these goals? 1. Transform tomato with ZAR1, JIM2, Roq1 and Bs2 We transformed two constructs into tomato. The first was construct 298 containing ZAR1 and JIM2. The second was construct 357 containing Roq1 and Bs2. Each construct was transformed into the elite inbred parent varieties TOM1 and TOM2. Cumulative with the previous reporting period, we have now obtained 75 plants for 298 transformed into TOM2 from 37 putative transformation events, 29 plants for 298 transformed into TOM1 from 14 putative transformation events, 127 plants for 357 transformed into TOM2 from 63 putative transformation events, and 15 plants for 357 transformed into TOM1 from 11 putative transformation events. Both TOM1 and TOM2 were more difficult to transform than the research variety used during Phase I (with TOM1 being particularly recalcitrant to transformation), and despite an intensive effort our transformation provider was unable to obtain the desired 100 independent transformation events for each combination. The obtained 125 putative independent events was only 31% of the desired 400. 2. Screen plants for resistance to Xanthomonas. 78 lines were subjected to phenotyping using the Xanthomonas growth assay. Lines excluded from the screen include ones that were not healthy enough for the assay, were sterile, and lines that received the NPTII gene but not the resistance gene cassette. 31 lines showed weak or no resistance and 47 showed moderate or strong resistance to Xanthomonas. 3. Screen the selected lines for desired "clean" insertion events by high-throughput sequencing A total of 127 plants were subjected to gDNA extraction, CAS9-enrichment and nanopore sequencing to characterize the insertion events. gDNA was extracted in pools of approximately six individual plants, with a total of 21 pools. The reads were analyzed by a custom bioinformatics pipeline to identify and characterize T-DNA insertions. Approximately 250 T-DNA insertion events were identified, but most were undesirable including tandem insertions, insertions of the NPTII selectable marker, or fragments of the resistance gene cassette. We identified only four unique clean insertion events. We used genotyping primers to confirm these events and match them back to individual plants. All four of these desired clean events were from the transformation of construct 298 into tomato variety TOM2. 4. Self plants and identify desired homozygous lines. We attempted to self the four plants with the desired clean resistance gene insertions. One plant was completely sterile and failed to produce any seeds. A second plant produced only four small seeds, all of which failed to germinate. This was unfortunate but not surprising, as approximately 30% of our transformed tomatoes are sterile. Presumably this is due to partial aneuploidy or chromosomal rearrangements that can occur during the tissue culture and transformation process. The third plant produced many seeds (~475) and the fourth produced 19 seeds. 5. Confirm disease resistance phenotype in the growth chamber. We grew up the progeny from the two plants with clean insertion events and tested them for resistance to Xanthomonas using a bacterial growth assay. The plants from one line had weak but consistent resistance whereas the plants from the other line had no detectable resistance. To better understand why so many of the transformants showed weak or no resistance despite being successfully transformed with the resistance gene cassette, we performed RNA-seq of some of the plants with strong, moderate, weak, and no resistance. We saw a correlation between strength of resistance gene expression and the strength of the resistance, with plants showing no or weak resistance having little or no expression of the resistance genes. Notably we used the native promoters for ZAR1 and JIM2 cloned from Solanum pennellii. Despite working well in the native context, these promoters do not seem to give consistently sufficient expression when transformed into tomato. This may be due to chromosomal position and local context in which the resistance gene cassette inserts into. 6-9. While we did obtain one tomato line with a clean insertion of ZAR1 and JIM2 which showed resistance to Xanthomonas, the resistance was rather weak at only about a 3-fold decrease in bacterial growth relative to the wild type. From our past work we know that we need to see at least a 10-fold decrease in bacterial growth, and ideally a 100-fold decrease, in order to see a qualitative disease resistance phenotype in the field. Therefore, we did not move forward with generating hybrid seed using this event.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Ahn, Y.J., Kim, H., Choi, S., Mazo?Molina, C., Prokchorchik, M., Zhang, N., Kim, B., Mang, H., Koehler, N., Kim, J., Lee, S., Yoon, H., Choi, D., Kim, M., Segonzac, C., Martin, G.B., Schultink, A., Sohn, K.H., 2023. Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities. New Phytologist, 239(5), pp.1935-1953.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:We published an academic paper describing some of our work on Xanthomonas resistance. This paper will primarily reach an academic audience but is also targetted toward those in industry looking for biotech solutions to control bacterial pathogens in tomato. Changes/Problems:The two major problems were difficulty in getting a sufficient number of transformants (we obtained only 125 putative independent events out of a desired 400) and insufficient expression of ZAR1 and JIM2 from the native promoters (two plants containing the desired ZAR1+JIM2 clean insertion event did not expressed the genes to a sufficient level to confer resistance to Xanthomonas). The transformation problem can be overcome in future work by better optimizing the transformation method for these tomato varieties or by using a variety that is more amenable to transformation and then introgressing the insertion into the desired background. Alternatively, a system that can more efficiently generate clean insertions could be used. The expression problem could be overcome by using non-native promoters that give stronger expression. The native promoters were selected because they work in Solanum pennellii, they have fewer regulatory and consumer acceptance concerns than using a strong viral promoter such as 35S. Additionally, ZAR1+JIM2 can give auto activity when highly expressed so we did not want promoters that give expression which is too strong. From Phase I we knew that the native promoters can work, which we also observed here, but unfortunately they only give moderate or strong resistance in about 50% of the transformants (and this could be skewed a by having multiple T-DNA insertions in a plant). Additionally, whereas making traditional transgenics with the desired gene of interest next to the selectable marker tends to select for insertion locations which favor expression of both the gene of interest and the selectable marker, our approach does not have this constraint. We are therefore able to recover plants that have the NPTII selectable marker inserted at a position that is favorable for gene expression whereas the gene of interest may be inserted at a different location that is not conducive for gene expression. What opportunities for training and professional development has the project provided? Nothing Reported 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? Nothing Reported

Impacts
What was accomplished under these goals? 1. Transform tomato with ZAR1, JIM2, Roq1 and Bs2 We transformed two constructs into tomato. The first was construct 298 containing ZAR1 and JIM2. The second was construct 357 containing Roq1 and Bs2. Each construct was transformed into the elite inbred parent varieties TOM1 and TOM2. Cumulative with the previous reporting period, we have now obtained 75 plants for 298 transformed into TOM2 from 37 putative transformation events, 29 plants for 298 transformed into TOM1 from 14 putative transformation events, 127 plants for 357 transformed into TOM2 from 63 putative transformation events, and 15 plants for 357 transformed into TOM1 from 11 putative transformation events. Both TOM1 and TOM2 were more difficult to transform than the research variety used during Phase I (with TOM1 being particularly recalcitrant to transformation), and despite an intensive effort our transformation provider was unable to obtain the desired 100 independent transformation events for each combination. The obtained 125 putative independent events was only 31% of the desired 400. 2. Screen plants for resistance to Xanthomonas. 78 lines were subjected to phenotyping using the Xanthomonas growth assay. Lines excluded from the screen include ones that were not healthy enough for the assay, were sterile, and lines that received the NPTII gene but not the resistance gene cassette. 31 lines showed weak or no resistance and 47 showed moderate or strong resistance to Xanthomonas. 3. Screen the selected lines for desired "clean" insertion events by high-throughput sequencing A total of 127 plants were subjected to gDNA extraction, CAS9-enrichment and nanopore sequencing to characterize the insertion events. gDNA was extracted in pools of approximately six individual plants, with a total of 21 pools. The reads were analyzed by a custom bioinformatics pipeline to identify and characterize T-DNA insertions. Approximately 250 T-DNA insertion events were identified, but most were undesirable including tandem insertions, insertions of the NPTII selectable marker, or fragments of the resistance gene cassette. We identified only four unique clean insertion events. We used genotyping primers to confirm these events and match them back to individual plants. All four of these desired clean events were from the transformation of construct 298 into tomato variety TOM2. 4. Self plants and identify desired homozygous lines. We attempted to self the four plants with the desired clean resistance gene insertions. One plant was completely sterile and failed to produce any seeds. A second plant produced only four small seeds, all of which failed to germinate. This was unfortunate but not surprising, as approximately 30% of our transformed tomatoes are sterile. Presumably this is due to partial aneuploidy or chromosomal rearrangements that can occur during the tissue culture and transformation process. The third plant produced many seeds (~475) and the fourth produced 19 seeds. 5. Confirm disease resistance phenotype in the growth chamber. We grew up the progeny from the two plants with clean insertion events and tested them for resistance to Xanthomonas using a bacterial growth assay. The plants from one line had weak but consistent resistance whereas the plants from the other line had no detectable resistance. To better understand why so many of the transformants showed weak or no resistance despite being successfully transformed with the resistance gene cassette, we performed RNA-seq of some of the plants with strong, moderate, weak, and no resistance. We saw a correlation between strength of resistance gene expression and the strength of the resistance, with plants showing no or weak resistance having little or no expression of the resistance genes. Notably we used the native promoters for ZAR1 and JIM2 cloned from Solanum pennellii. Despite working well in the native context, these promoters do not seem to give consistently sufficient expression when transformed into tomato. This may be due to chromosomal position and local context in which the resistance gene cassette inserts into. 6-9. While we did obtain one tomato line with a clean insertion of ZAR1 and JIM2 which showed resistance to Xanthomonas, the resistance was rather weak at only about a 3-fold decrease in bacterial growth relative to the wild type. From our past work we know that we need to see at least a 10-fold decrease in bacterial growth, and ideally a 100-fold decrease, in order to see a qualitative disease resistance phenotype in the field. Therefore, we did not move forward with generating hybrid seed using this event.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Ahn, Y.J., Kim, H., Choi, S., Mazo?Molina, C., Prokchorchik, M., Zhang, N., Kim, B., Mang, H., Koehler, N., Kim, J., Lee, S., Yoon, H., Choi, D., Kim, M., Segonzac, C., Martin, G.B., Schultink, A., Sohn, K.H., 2023. Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities. New Phytologist, 239(5), pp.1935-1953.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems:The major problem we had during this reporting period was difficulty in getting a sufficient number of tomato transformants. The two elite inbred tomato lines we are working with, TOM1 and TOM2, are much more difficult to transform than the research variety we worked with during Phase I. To help overcome this, we provided a very large number of seeds to the transformation provider to allow them to scale up the transformation experiments. Other problems we encountered were the low frequency of clean insertion events and not all transformants showing good resistance to Xanthomonas (presumably from differences in expression level between the transformants). This was somewhat expected based on our Phase I results and is the reason why we need several hundred transformation events to find the desired clean insertion events with good expression. We are continuing to look in Ptr1 gene as a additional Xanthomonas resistance gene to use in tomato. The desirable aspect of Ptr1 is that, like ZAR1 and JIM2, it is from a sexually compatible tomato species and therefore subject to less regulatory and consumer acceptance concerns than Roq1 and Bs2. What opportunities for training and professional development has the project provided? Nothing Reported 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?We will continue to obtain more tomato transformants. These plants will be screened by phenotyping and genotyping to obtain the desired clean insertion events that confer strong resistance to Xanthomonas.

Impacts
What was accomplished under these goals? 1. Transform tomato with ZAR1, JIM2, Roq1, Bs2 We received two elite inbred tomato lines from a collaborating seed company and sent the seeds and constructs to our service provider for tomato transformation. We propagated more seeds for these two parent lines to be able to provide more seed to the service provider. In total we sent several thousand seeds for the two varieties to be transformed. There were two constructs to be transformed into tomato. The first was construct 298 containing ZAR1 and JIM2. The second was construct 357 containing Roq1 and Bs2. Each construct was transformed into both the parent varieties (TOM1 and TOM2). As of 8-31-2022 we obtained 60 plants for construct 298 transformed into TOM2 from 29 putative transformation events, 18 plants for construct 298 transformed into TOM1 from 10 putative transformation events, 68 plants for construct 357 transformed into TOM2 from 42 putative transformation events, and 2 plants for construct 357 transformed into TOM1 from 1 putative transformation event. Notably, both TOM1 and TOM2 proved to be more difficult to transform than the research variety used during Phase I, with TOM1 being particularly difficult. Our goal was to get 100 independent transformation events for transformation. Our provider is continuing to try to get more transformants while we moved forward with analyzing the lines we have. 2. Screen the plants for resistance to Xanthomonas The transformed plants (T0 generation) were tested for resistance to Xanthomonas using a bacterial growth assay. As of 8-31-2022, 19 plants showed moderate or strong resistance to Xanthomonas, whereas 11 showed no or weak resistance. Testing is in progress on the remaining plants. 3. Screen the selected lines for desired "clean" insertion events by high-throughput sequencing We genotyped the plants and found that not all were successfully transformed with the desired transgene (in some cases they received NPTII but not the desired resistance gene cassette). Additionally, some plants did not do well after transfer to soil (typically they these plants looked unhealthy before transfer with poor rooting), and some other plants appeared healthy but did not produce any viable seeds (likely due to partial aneuploidies induced during the tissue culture and transformation process). Only plants that tested positive for the resistance gene cassette and appeared healthy were used for sequencing. We developed a new protocol to characterize the insertion events in the tomato plants. Rather than using an Illumina based approach, as in Phase I, we used a CAS9-enrichment followed by nanopore sequencing. The nanopore sequencing worked quite well and gave us results in a couple days as opposed to a turn around of 3-6 weeks by sending DNA off for Illumina sequencing. Initially the quality of the data was poor, but by improving the DNA purification protocol to get cleaner genomic DNA we were able to get good reads and sufficient enrichment of the target DNA. We used the nanopore protocol to screen plants for the desired clean insertion events. So far 20 plants have been sequenced and 55 insertion events identified with the sequencing (averaging 2.5 insertion events per plant). This includes both NPTII insertions and resistance gene cassette insertions. Most insertion events were complex / tandem insertions, in which multiple T-DNAs were detected adjacent to each other. 8 events had one border that was cleanly inserted into the tomato genome but none had both borders clean. A low efficiency of clean insertions was expected based on our Phase I results and this is why we wanted to get a larger number of transgenic lines. 4. Self plants and identify the desired homozygous lines. Although we didn't identify any clean insertion events in plants with strong resistance to Xanthomonas yet, we did start selfing some of the plants that showed at least moderate resistance to Xanthomonas. We have seed from six transformants, with approximately 40 primary transgenic plants in the greenhouse. 5-9: Not yet started. In addition to the above, we have been investigating an additional Xanthomonas resistance gene from the plant Solanum lycopersicoides with two collaborating academic labs. This gene, Ptr1, mediates perception of the Xanthomonas effector AvrBsT and is required for strong resistance to Xanthomonas perforans triggered by this avirulence gene in Solanum lycopersicoides and Nicotiana benthamiana. We found that tomato transformed with Ptr1 did not have any resistance to Xanthomonas. This, along with some other data, suggested the involvement of an additional gene in the AvrBsT recognition pathway. We attempted to identify this additional gene by a mapping-based approach using a Solanum lycopersicoides x Solanum lycopersicum mapping population. Our results indicated that either an additional gene required for Ptr1 activity is not required, that the such a gene already exists in tomato, or that such a gene acts as negative regulator of Ptr1. We did an RNA-seq of the tomato plant transformed with Ptr1 and found no detectable expression of the Ptr1 transgene, possibly explaining the lack of disease resistance phenotype without the need for an additional component. Knowing that Ptr1 has some autoactivity when transiently overexpressed in Nicotiana benthamiana, we did a reverse genetic screen and identified two closely related genes, which we named R4H-1 and R4H-6, which act as negative regulators of Ptr1. Domesticated tomato has an apparently functional copy of this negative regulator, suggesting that it does not need to be added to have an appropriately functional Ptr1. Overall, these investigations suggest that the failure of the Ptr1 transgenic tomato to have resistance to Xanthomonas is probably due to a lack of Ptr1 expression in the line transformed with Ptr1.

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:We had discussions with several tomato seed companies regarding our bacterial disease resistance traits. We signed an agreement with one company to move the traits into an elite tomato variety. Changes/Problems:The major problem we had during this period was that a planned collaborator decided not to move forward and it took us a long time to get a new collaboration negotiated with a different company. We finally got this completed in August 2020 and are now moving forward with the transformations. Because we were not able to move forward with the transformations, we tested out an additional resistance gene that we discovered in collaboration with two academic labs. This resistance gene recognizes the AvrBsT effector protein from Xanthomonas perforans and confers resistance to this disease in tomato. We tested transient and stable expression of this gene. The gene is from a wild tomato species that is sexually compatible with tomato. This is desireable because it is subject to less regulation and less market scrutiny than genes from more distant relatives (such as the Roq1 gene from tobacco). We obtained a tomato introgression line with the gene and found it to be highly resistant to Xanthomonas (nearly as resistant as our tomato line containing ZAR1, JIM2 and Roq1). We also obtained a stable transgenic tomato line expressing the gene but found that it was not resistant to Xanthomonas. Based on this and other data we now beleive that there is a second gene that is part of the pathway and we are exploring several candidates for this second component. What opportunities for training and professional development has the project provided? Nothing Reported 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?The project will continue mostly as planned although delayed as we were delayed in getting the tomato seeds. We now have the seeds and the transformations are underway. Due to the delay we spent less than planned during the first year and we will likely file a no cost extension next year to have time to complete the project.

Impacts
What was accomplished under these goals? We had several setbacks during this period. The first was that one tomato seed company decided not to move forward with a collaboration. We signed a collaboration agreement with a different company, but it then took several months to negotiate it and an additional couple months to get the seed for the line. While waiting for the seeds we: - Made a new version of the Roq1-Bs2 construct with improved activity - Found an additional Xanthomonas resistance trait (in collaboration with two academic labs) and tested it by transient expression, stable expression and stable knockout in the source plant. - Developed a plan for applying for a Regulator Status Review with the USDA We did not want to move forward engineering the tomato seeds until we had access to an elite variety. We therefore put the transformations on hold until we obtained the seeds. The transformations are currently underway to complete objective 1, with planned completion in early 2022.

Publications