Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains

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1. 

   Version published to 10.7554/elife.84864 on eLife

   Jun 20, 2023
2. 

   eLife

   May 25, 2023

   Author Response

   Reviewer #1 (Public Review):

   Luu et al. have developed a genome-edited African elite rice variety, Komboka. The work was initiated in response to the outbreak in Eastern Africa by Xanthomonas oryzae strains that are phylogenetically related to Asian strains and carry TALes, similar to strains from China, possessing an expanded repertoire of TALes compared to those in endemic strains. As these emerging strains contain TALe targeting SWEET11a, as well as that suppressing Xa1, pthXo1, and iTALes, the authors have generated edited lines targeting promoter regions of SWEET11a, 13 and 14 in African elite rice variety, Komboka. The same team has previously generated genome-edited lines targeting the promoter regions of SWEET11a, 13, and 14 in varieties Kitaake, IR64, and Ciherang-Sub1. Bacterial blight outbreaks and emerging …

   Author Response

   Reviewer #1 (Public Review):

   Luu et al. have developed a genome-edited African elite rice variety, Komboka. The work was initiated in response to the outbreak in Eastern Africa by Xanthomonas oryzae strains that are phylogenetically related to Asian strains and carry TALes, similar to strains from China, possessing an expanded repertoire of TALes compared to those in endemic strains. As these emerging strains contain TALe targeting SWEET11a, as well as that suppressing Xa1, pthXo1, and iTALes, the authors have generated edited lines targeting promoter regions of SWEET11a, 13 and 14 in African elite rice variety, Komboka. The same team has previously generated genome-edited lines targeting the promoter regions of SWEET11a, 13, and 14 in varieties Kitaake, IR64, and Ciherang-Sub1. Bacterial blight outbreaks and emerging pathogen lineages remain to be a threat to rice production. Thus, efforts in targeting pathogen weaknesses to generate genome-edited varieties possessing broad-spectrum resistance are required. The survey, collection of isolates, and strain characterization studies on >800 strains are commendable. This study has taken advantage of this ongoing collection to stay ahead in the arms race to deploy broad-spectrum resistance in an elite rice variety using TALe targets.

   Overall conclusions presented here are supported to some extent; however, I have listed some of my comments and concerns below.

   1. Data in supplementary table 2 suggests that Komboka is still a moderately resistant variety under field conditions in Africa, with a disease severity scale of 2 i.e. 4-6% disease severity, compared to other varieties having a disease severity scale of 5. Thus, I am not convinced that emerging strains are of concern on the Komboka variety under field conditions, thus, question the justification of Komboka being a choice for editing to tackle emerging strains.

   We apologize, because the Table 2 is admittedly hard to read with the geo data. We have thus added a new figure 1 with maps. Please note that the data in this Table are from 2022. If you look at for example the Morogoro region (Dakawa and Lunkege), it appears that also there, the initial scale (number of plants infected) was low and became more severe in the subsequent years as one might expect. We thus hypothesize, that in the upcoming analyses, the scale will also become much higher, thus this snapshot cannot serve as a measure of general susceptibility. As we noted in the response to the Editor, the Kaufmann clipping assays are widely used by breeders to evaluate resistance in greenhouse conditions, and since the assays uses severe wounding and extremely high bacterial inocula, this assay is a reliable measure of susceptibility. Note also, that Komboka was chosen before the outbreak was characterized. Our data show that Komboka is highly susceptible to Asian strains, as well as to the introduced strains. Note also that we characterized the R gene outfit as far as feasible, an found two R genes that can explain the resistance to the endemic African strains. Note that single, double and triple R gene mutant combinations have been broken in India, thus we deemed it necessary to create a rational approach that prevents SWEET gene recruitment to generate broad spectrum resistance. xa13 has likely only been broken by circumventing SWEET11a (by using SWEET13 or 14), but still stands up in quintuple breeding combinations in India. Thus, we expect that our lines will be rather robust, which will have to be tested in future field trials in Kenya where this variety is highly cultivated. We added text to Results, Discussion sections and a new section on sampling in Methods with respective references that show the correlation of data from assays with the same strains in greenhouse and field.

   1. Is Xa4 from Komboka related to Xa4_Teqing? The breakdown of Xa4T was due to the mutant allele of avrXa4 in virulent Xoo CR6. But this breakdown was accompanied by a fitness penalty and residual QTL had a significant residual effect on virulent strains. Would this be why Komboka carrying Xa1 (Xa45(t) and Xa4 under field conditions still showed moderate resistance? But Xoo strains showed susceptibility in leaf clipping assays.

   We apologize, this was a typo that has been corrected. Komboka is a high yielding variety, we thus cannot comment on any yield penalty here, it is superior and widely accepted now in Kenya. And we responded regarding on the moderate resistance in the previous paragraph. Komboka is fully susceptible to the Asian strains that induce SWEET11a.

   1. I felt a bit of a disconnect in sections on phenotypic assays confirming the virulence profile of strains on Komboka and then understanding mechanisms underlying virulence since the same strains used in path data were not the ones mentioned in WGS and TALe analysis, leaving the readers with the only one strain to support the hypothesis of the basis for higher disease severity on Komboka due to new TALes, pthXo1, and iTALe. Do authors have pathogenicity data for African strains T19, Dak16, and Xoo3-1 that grouped with endemic African strains on Komboka? Authors present data on CIX4457, 4458, and 4462 being virulent on Komboka, and show that they cluster with Asian strains. However, in the tree, 4462 is the only one shown to be closely related to Chinese strains. Where are 4457 and 4458 placed? Do 4457 and 4458 also contain pthXo1 and iTALe? Authors could also provide path data for 4506/4509 that they included in TALe figure and in the phylogenetic tree.

   We had initiated WGS of 8 strains (3 from Dakawa and 5 from Lukenge), but at the time of submission, not all genomes were fully polished. Although not all are in a publishable state by now, we were able to determine the similarity as well as presence of pthXo1 and iTALes. The number of SNPs among the 8 strains is extremely low (between 1 and 4), strongly intimating that they are siblings. They are so similar, that we can at present not trace the origin. All eight strains isolated in Dakawa in 2019 and in Lukenge in 2021 contain iTALes and the PthXo1B variant. With near certainty that they are all derived from a single introduction event. We fully understand your comment. We apologize, since we should not have used the CIX nomenclature, which was introduced to obtain a more reliable code for the strains. We have introduced a clearer nomenclature while keeping the code for the database. We added a new Figure 2-supplement 1 which shows that Komboka is susceptible not only to the three strains isolated in Dakawa in 2019, but also to one of the strains isolated from Lukenge in 2021. We replaced Fig. 3 with a new phylogenetic tree including the eight strains and provide more detailed information on the relation of those strains. In principle it would be sufficient to use a single isolate in this case. We now provide, as far as possible the new data (analysis is ongoing) as well as new data for some strains collected in 2022 and conclude that also the strains identified in 2022 are derivatives from an initial introduction in the Morogoro region. It is also clear from Fig. 2 and supplement that Komboka is fully susceptible to the strains isolated from Dakawa and Lukenge, as susceptible as to the Philippine reference strain PXO99A, which also uses PthXo1.

   1. The authors present pathogenicity data on EBE-edited T0, T1, and T2 lines of Komboka which are promising against the Tanzanian strains carrying new TALes. The cas9/cpf1 system developed here to target multiple EBEs will be a valuable contribution to the scientific community. What are the agronomic characteristics of these edited lines? As the edited lines have not been tested against a diversity panel of Asian and African strains, I would be skeptical of the choice of the term "broad-spectrum" yet.

   Virulence of Xoo depends critically on the recruitment of at least one of the three SWEETs (11a, 13 or 14). Single R genes, such as xa13 can be overcome by using SWEET13 or 14. All strains that are virulent carry at least one TALe that targets a SWEET. Thus, by blocking all known EBEs, we obtain broad spectrum resistance. We have not observed a single case yet where this is not working. Note that in the case of EBE edited Kitaake, we tested about 100 different strains from a world-wide collection, for IR64 and Ciherang-Sub 1 also many representative strains, and we now show data for Komboka and additional varieties. Thus, based on the current knowledge, including the information gained from Xoo genome sequences that have been published, e.g., recently from India, there is at present no strain known that can overcome this resistance.

   Regardless of my comment earlier on Komboka being moderately resistant under field conditions and thus a questionable choice for EBE-editing here, the genome-edited lines in any variety imparting resistance to bacterial blight remain to be a valuable contribution to managing disease outbreaks.

   We commented on the interpretation of moderate resistance above, but appreciate the comment that these lines will be valuable.

   1. As this manuscript utilizes the diversity of African strains to generate edited lines, it would be good to make diagnostics and path data for 833 strains available to the scientific community (instead of select strains as indicated in the supplementary table), especially for the fact stated here in the manuscript about scarce data on Xoo in Africa and the goal of systematic comparison of the pathogen population.

   We are currently preparing a manuscript that will include an extensive analysis of these strains, and focus on the diversity of African Xoo strains, i.e., MLVA-based diversity of the collection. This manuscript, which is in preparation, will include the requested data.

   Reviewer #2 (Public Review):

   This study describes the emergence of virulent strains of the rice bacterial blight pathogen Xanthomonas oryze pv. oryzae in the Morogoro rice-growing region in Tanzania. The aims of the study were to describe the virulence features of the emerging population, as compared to previous bacterial blight outbreaks in Africa, and generate an elite rice variety that is resistant to both pathogen populations. To achieve these aims, the authors characterized the genetic basis of the virulence of these new strains by sequencing the genomes of three representative strains and phenotyping using excellent genetic resources for identifying the susceptibility gene targets of this pathogen in rice. They then used two rounds of hybrid CRISPR-Cas9/Cpf1 to successfully edit six targets of the pathogen in an East African rice variety, which conferred resistance to all strains tested.

   The strengths of this paper are the systematic analysis of the virulence of emerging pathogen strains relative to strains from previous outbreaks and the successful creation of edited lines that will form the basis for continued efforts to gain regulatory approval for the introduction of resistant rice in East Africa. The creation of the edited line is a substantial and important contribution, indeed, the authors include strains collected in 2021 and include disease severity data from 2022 in the supplementary data, illustrating the urgent need for solutions.

   The weaknesses of the study are largely related to the quick turnaround between data collection and manuscript submission.

   1. Different strains are used for different experimental work and sequence analysis, making relationships between different parts of the work unclear and also more challenging for the reader to follow because of changing strain designations. CIX4457, CIX4458, and CIX4462 were virulent on rice near-isogenic-lines, CIX4457 and CIX4505 were used for identifying SWEET targets and phenotyping edited lines, while whole genome sequencing was conducted with CIX4462, CIX4506, CIX4509.

   We added new information which demonstrates that the strains isolated in 2019 in Dakawa and the strains from Lukenge (2021) are very closely related and differ only by a 1 to 4 core genome SNPs (see new supp Fig. 3A). We added a new Figure2-supplementary Figure 1 and expanded Table 1 to show that the strains from Lukenge and Dakawa behave in a similar manner. We are aware of the differences in the figures but hopefully have now addressed them in an acceptable manner, we did not want to combine data from different experiments. The differences in strain use are due to i) the different timing of strains sampling and isolation (those from 2019 were isolated first and the long and tedious work of leaf-clipping the whole set of NILs with all the diversity strain panel did therefore not include Tanzanian strains from 2021 that were isolated much later also due to long delay in having the infected leaf material sent out; including them in the NILs testing would have taken us another year given the volume of this experiment), and ii) the variable quality of whole genome sequencing of the strains. Overall, we have sequenced the genome of 8 newly introduced strains including 3 from Dakawa_2019 and 5 from Lukenge_2021 (see new suppl. Table 3 that gives a detailed overview of the genomic analysis of these strains). The best genome sequences were obtained for strains CIX4462, CIX4506 and CIX4509 (renamed in the revised version of this MS and for sake of clarity as iTzDak19-3, iTzLuk21-1 and iTzLuk21-2) of which a circularized chromosome could be generated. Unfortunately, these were not the strains that we had selected for SWEET characterization and phenotyping of edited lines, whereby one representative strain of each collection had been randomly picked, namely CIX4457 and CIX4505 (now iTzDak19-1 and iTzLuk21-3, respectively). To reconcile these two sets of data and show that strains from Dakawa and Lukenge are actually extremely similar, we have performed a SNP-based phylogenetic analysis of the 8 strains demonstrating that they all cluster as one homogeneous genetic lineage, in line with a scenario whereby all these strains result of a single introduction event from Asia. Careful analysis of these additional genomes also confirmed the presence of a pthXo1like allele (pthXo1B) and iTALes in all Tanzanian strains introduced from Asia. One exception is strain iTzLuk21-3 (CIX4505) where the poor quality of the pthXo1B sequence with potential frameshifts prevented any confirmatory analysis. Taken together, these data support the hypothesis that all new isolates, irrespectively of the year of sampling, are genetically very close and share the same virulence characteristics.

   1. Disease survey results from 2022 are listed in Supplementary Table 2, but it is challenging for the reader to summarize across many lines of data, which appear to represent individual samples.

   We agree that this was not the best way to show the data. In addition to the new suppl. Tables 1 and 3 we have now generated a new Figure 1 which contains maps of the disease distribution and severity across Tanzania in the different years as well as photos from the fields in Dakawa from 2019 and Lukenge in 2021 that highlight the massive infections.

   1. The focus of the editing is Komboka but bacterial blight in 2022 was mostly on other varieties. It would be helpful to have more context on this variety and what has prevented adoption by the growers in the Morogoro region to date.

   The variety was chosen several years ago after extensive consultations with breeders from IRRI, IRRI Africa, and India, since it is high yielding, and was specifically generated for Kenya where it has a high level of adoption. Tanzania has apparently not yet adopted this variety as you can see from Table 2. Also, Tanzania does NOT have any regulations for genome edited crops and we can thus NOT provide the lines to Tanzania. By contrast, Kenya has established a regulatory framework by which the local government authorities can import transgene-free edited lines. We are currently segregating the transgenes out and have established a through set of measures to validate whether the lines still contain transgenes (including vector backbone and T-DNA remnants). Tanzania will have to establish suitable guidelines. We would like to note that establishing protocols for different elite varieties is challenging and time consuming and we had early on, in 2019, decided to initiate work on transformation protocols for this variety. If Tanzania also adopt regulations, it would be possible to provide the lines to Tanzania as well, and possibly by then Tanzania has a higher level of adoption of Komboka. If you look at the maps we show, it is very likely that the disease will spread to all neighboring countries, including Kenya. Thus, our lines may become one possible measure to try to address the outbreak.

   Reviewer #3 (Public Review):

   One key finding of this work is the identification of Xanthomonas oryzae pv. oryzae (Xoo) strains in Africa, based on their genomes sequence and their TALE repertoires, have high similarity with Asian strains. Asian Xoo strains typically overcome NLR-mediated recognition of TALEs in rice by so-called iTALEs. Moreover, some Asian strains contain a TALE resembling PthXo1, a TALE protein that was shown to overcome xa5 resistance.

   The authors now found that some of the newly identified African strains have iTALEs and PthXo1-like TALEs. Such newly evolved African strains were found to be fully virulent on the African rice elite variety Komboka, which is resistant to a broad panel of African Xoo strains.

   Previous studies have shown that TALEs bind to effector binding elements (EBEs) present in promoters of rice SWEET genes to promote disease. Work from the lab of the authors and other labs has shown that TALEs can no longer promote the disease if matching EBEs are changed or deleted by CRISPR or TALEN-mediated mutagenesis. In fact, pioneering work by Bing Yang, one of the authors of this article published about ten years ago a Nature Biotechnology article where he showed that rice plants with mutated EBEs are resistant to Xoo. Recently, a combined effort of the Yang and Frommer labs resulted in two further Nature Biotechnology publications (2019), in which they described along with other useful tools rice lines where multiple EBEs were mutagenized in parallel and that provide broad spectrum resistance.

   The work under review describes now CRISPR mutagenesis of an African elite cultivar resulting in a line that mediates resistance to Asian and newly evolved African strains.

   Overall, the work is technically sound. Yet, the approach that has been described - mutagenesis of multiple EBEs - has been used before and is a routine procedure for labs that are focused on such undertakings. While such approaches do not provide new insights for fundamental research, they nevertheless are certainly important and useful in translational research, as demonstrated here.

   We thank reviewer for the comments. If we may, we would like to add aspects of novelty. We detected an outbreak that is spreading. We determined the disease mechanism, and we used CPF1 to obtain ‘optimal’ mutations at all sites (massive improvement over 2019 publication, which used Cas9) and we try to provide a solution for the outbreak when it spreads to Kenya, or when Tanzania and neighboring Countries adopt similar guidelines. This seems highly urgent das Reviewer 2 points out.

   Read the original source
3. 

   eLife

   May 25, 2023

   eLife assessment

   This valuable study shows that new, virulent genotypes of Xanthomonas oryze pv. oryzae, that are similar to strains present in east Asia, cause outbreaks of bacterial blight of rice in Tanzania. The authors' use of CRISPR-based gene editing on multiple pathogen targets in an elite African rice variety to create lines resistant to both endemic and emerging pathogen strains in Africa makes for a compelling contribution to meet this alarming development. The work describing the new strains of the pathogen is solid but could be stronger if there were genome sequence data for all strains examined and a clearer presentation of recent disease outbreaks and their severity.

   Read the original source
4. 

   eLife

   May 25, 2023

   Reviewer #1 (Public Review):

   Luu et al. have developed a genome-edited African elite rice variety, Komboka. The work was initiated in response to the outbreak in Eastern Africa by Xanthomonas oryzae strains that are phylogenetically related to Asian strains and carry TALes, similar to strains from China, possessing an expanded repertoire of TALes compared to those in endemic strains. As these emerging strains contain TALe targeting SWEET11a, as well as that suppressing Xa1, pthXo1, and iTALes, the authors have generated edited lines targeting promoter regions of SWEET11a, 13 and 14 in African elite rice variety, Komboka. The same team has previously generated genome-edited lines targeting the promoter regions of SWEET11a, 13, and 14 in varieties Kitaake, IR64, and Ciherang-Sub1. Bacterial blight outbreaks and emerging pathogen lineages …

   Reviewer #1 (Public Review):

   Luu et al. have developed a genome-edited African elite rice variety, Komboka. The work was initiated in response to the outbreak in Eastern Africa by Xanthomonas oryzae strains that are phylogenetically related to Asian strains and carry TALes, similar to strains from China, possessing an expanded repertoire of TALes compared to those in endemic strains. As these emerging strains contain TALe targeting SWEET11a, as well as that suppressing Xa1, pthXo1, and iTALes, the authors have generated edited lines targeting promoter regions of SWEET11a, 13 and 14 in African elite rice variety, Komboka. The same team has previously generated genome-edited lines targeting the promoter regions of SWEET11a, 13, and 14 in varieties Kitaake, IR64, and Ciherang-Sub1. Bacterial blight outbreaks and emerging pathogen lineages remain to be a threat to rice production. Thus, efforts in targeting pathogen weaknesses to generate genome-edited varieties possessing broad-spectrum resistance are required. The survey, collection of isolates, and strain characterization studies on >800 strains are commendable. This study has taken advantage of this ongoing collection to stay ahead in the arms race to deploy broad-spectrum resistance in an elite rice variety using TALe targets.

   Overall conclusions presented here are supported to some extent; however, I have listed some of my comments and concerns below.

   1. Data in supplementary table 2 suggests that Komboka is still a moderately resistant variety under field conditions in Africa, with a disease severity scale of 2 i.e. 4-6% disease severity, compared to other varieties having a disease severity scale of 5. Thus, I am not convinced that emerging strains are of concern on the Komboka variety under field conditions, thus, question the justification of Komboka being a choice for editing to tackle emerging strains.

   2. Is Xa4 from Komboka related to Xa4_Teqing? The breakdown of Xa4T was due to the mutant allele of avrXa4 in virulent Xoo CR6. But this breakdown was accompanied by a fitness penalty and residual QTL had a significant residual effect on virulent strains. Would this be why Komboka carrying Xa1 (Xa45(t) and Xa4 under field conditions still showed moderate resistance? But Xoo strains showed susceptibility in leaf clipping assays.

   3. I felt a bit of a disconnect in sections on phenotypic assays confirming the virulence profile of strains on Komboka and then understanding mechanisms underlying virulence since the same strains used in path data were not the ones mentioned in WGS and TALe analysis, leaving the readers with the only one strain to support the hypothesis of the basis for higher disease severity on Komboka due to new TALes, pthXo1, and iTALe. Do authors have pathogenicity data for African strains T19, Dak16, and Xoo3-1 that grouped with endemic African strains on Komboka? Authors present data on CIX4457, 4458, and 4462 being virulent on Komboka, and show that they cluster with Asian strains. However, in the tree, 4462 is the only one shown to be closely related to Chinese strains. Where are 4457 and 4458 placed? Do 4457 and 4458 also contain pthXo1 and iTALe? Authors could also provide path data for 4506/4509 that they included in TALe figure and in the phylogenetic tree.

   4. The authors present pathogenicity data on EBE-edited T0, T1, and T2 lines of Komboka which are promising against the Tanzanian strains carrying new TALes. The cas9/cpf1 system developed here to target multiple EBEs will be a valuable contribution to the scientific community. What are the agronomic characteristics of these edited lines? As the edited lines have not been tested against a diversity panel of Asian and African strains, I would be skeptical of the choice of the term "broad-spectrum" yet.
   Regardless of my comment earlier on Komboka being moderately resistant under field conditions and thus a questionable choice for EBE-editing here, the genome-edited lines in any variety imparting resistance to bacterial blight remain to be a valuable contribution to managing disease outbreaks.

   5. As this manuscript utilizes the diversity of African strains to generate edited lines, it would be good to make diagnostics and path data for 833 strains available to the scientific community (instead of select strains as indicated in the supplementary table), especially for the fact stated here in the manuscript about scarce data on Xoo in Africa and the goal of systematic comparison of the pathogen population.

   Read the original source
5. 

   eLife

   May 25, 2023

   Reviewer #2 (Public Review):

   This study describes the emergence of virulent strains of the rice bacterial blight pathogen Xanthomonas oryze pv. oryzae in the Morogoro rice-growing region in Tanzania. The aims of the study were to describe the virulence features of the emerging population, as compared to previous bacterial blight outbreaks in Africa, and generate an elite rice variety that is resistant to both pathogen populations. To achieve these aims, the authors characterized the genetic basis of the virulence of these new strains by sequencing the genomes of three representative strains and phenotyping using excellent genetic resources for identifying the susceptibility gene targets of this pathogen in rice. They then used two rounds of hybrid CRISPR-Cas9/Cpf1 to successfully edit six targets of the pathogen in an East African rice …

   Reviewer #2 (Public Review):

   This study describes the emergence of virulent strains of the rice bacterial blight pathogen Xanthomonas oryze pv. oryzae in the Morogoro rice-growing region in Tanzania. The aims of the study were to describe the virulence features of the emerging population, as compared to previous bacterial blight outbreaks in Africa, and generate an elite rice variety that is resistant to both pathogen populations. To achieve these aims, the authors characterized the genetic basis of the virulence of these new strains by sequencing the genomes of three representative strains and phenotyping using excellent genetic resources for identifying the susceptibility gene targets of this pathogen in rice. They then used two rounds of hybrid CRISPR-Cas9/Cpf1 to successfully edit six targets of the pathogen in an East African rice variety, which conferred resistance to all strains tested.

   The strengths of this paper are the systematic analysis of the virulence of emerging pathogen strains relative to strains from previous outbreaks and the successful creation of edited lines that will form the basis for continued efforts to gain regulatory approval for the introduction of resistant rice in East Africa. The creation of the edited line is a substantial and important contribution, indeed, the authors include strains collected in 2021 and include disease severity data from 2022 in the supplementary data, illustrating the urgent need for solutions.

   The weaknesses of the study are largely related to the quick turnaround between data collection and manuscript submission.
   (1) Different strains are used for different experimental work and sequence analysis, making relationships between different parts of the work unclear and also more challenging for the reader to follow because of changing strain designations. CIX4457, CIX4458, and CIX4462 were virulent on rice near-isogenic-lines, CIX4457 and CIX4505 were used for identifying SWEET targets and phenotyping edited lines, while whole genome sequencing was conducted with CIX4462, CIX4506, CIX4509.
   (2) Disease survey results from 2022 are listed in Supplementary Table 2, but it is challenging for the reader to summarize across many lines of data, which appear to represent individual samples.
   (3) The focus of the editing is Komboka but bacterial blight in 2022 was mostly on other varieties. It would be helpful to have more context on this variety and what has prevented adoption by the growers in the Morogoro region to date.

   Read the original source
6. 

   eLife

   May 25, 2023

   Reviewer #3 (Public Review):

   One key finding of this work is the identification of Xanthomonas oryzae pv. oryzae (Xoo) strains in Africa, based on their genomes sequence and their TALE repertoires, have high similarity with Asian strains. Asian Xoo strains typically overcome NLR-mediated recognition of TALEs in rice by so-called iTALEs. Moreover, some Asian strains contain a TALE resembling PthXo1, a TALE protein that was shown to overcome xa5 resistance.

   The authors now found that some of the newly identified African strains have iTALEs and PthXo1-like TALEs. Such newly evolved African strains were found to be fully virulent on the African rice elite variety Komboka, which is resistant to a broad panel of African Xoo strains.

   Previous studies have shown that TALEs bind to effector binding elements (EBEs) present in promoters of rice …

   Reviewer #3 (Public Review):

   One key finding of this work is the identification of Xanthomonas oryzae pv. oryzae (Xoo) strains in Africa, based on their genomes sequence and their TALE repertoires, have high similarity with Asian strains. Asian Xoo strains typically overcome NLR-mediated recognition of TALEs in rice by so-called iTALEs. Moreover, some Asian strains contain a TALE resembling PthXo1, a TALE protein that was shown to overcome xa5 resistance.

   The authors now found that some of the newly identified African strains have iTALEs and PthXo1-like TALEs. Such newly evolved African strains were found to be fully virulent on the African rice elite variety Komboka, which is resistant to a broad panel of African Xoo strains.

   Previous studies have shown that TALEs bind to effector binding elements (EBEs) present in promoters of rice SWEET genes to promote disease. Work from the lab of the authors and other labs has shown that TALEs can no longer promote the disease if matching EBEs are changed or deleted by CRISPR or TALEN-mediated mutagenesis. In fact, pioneering work by Bing Yang, one of the authors of this article published about ten years ago a Nature Biotechnology article where he showed that rice plants with mutated EBEs are resistant to Xoo. Recently, a combined effort of the Yang and Frommer labs resulted in two further Nature Biotechnology publications (2019), in which they described along with other useful tools rice lines where multiple EBEs were mutagenized in parallel and that provide broad spectrum resistance.

   The work under review describes now CRISPR mutagenesis of an African elite cultivar resulting in a line that mediates resistance to Asian and newly evolved African strains.

   Overall, the work is technically sound. Yet, the approach that has been described - mutagenesis of multiple EBEs - has been used before and is a routine procedure for labs that are focused on such undertakings. While such approaches do not provide new insights for fundamental research, they nevertheless are certainly important and useful in translational research, as demonstrated here.

   Read the original source
7. 

   Version published to 10.1101/2022.11.20.517251v1 on bioRxiv

   Nov 22, 2022