Periodic Reporting for period 4 - ACHILLES-HEEL (Crop resistance improvement by mining natural and induced variation in host accessibility factors)
Okres sprawozdawczy: 2020-03-01 do 2022-02-28
Increasing crop yield to feed the world is a grand challenge of the 21st century but it is hampered by diseases caused by filamentous plant pathogens. The arms race between pathogen and plant demands constant adjustment of crop germplasm to tackle emerging pathogen races with new virulence features. To date, most crop disease resistance has relied on specific resistance genes that are effective only against a subset of races.
We cannot solely rely on classical resistance genes to keep ahead of the pathogens. There is an urgent need to develop approaches based on knowledge of the pathogen’s Achilles heel: core plant processes that are required for pathogen colonization.
We reasoned that disease resistance based on manipulation of host accessibility processes has a higher probability for durability, and is best identified using a broad host-range pathogen. Thus, we employed the filamentous pathogen Phytophthora palmivora to mine plant alleles and unravel host processes providing microbial access in roots and leaves of monocot and dicot plants.
In Aim 1 we have utilized plant symbiosis mutants and allelic variation to elucidate general mechanisms of colonization by filamentous microbes. This enabled us to identify genes which help creating an environment conducive to infection by filamentous pathogens. Removal of these genes renders specific plant tissues more resilient to infection. The identified genes can thus be considered susceptibility genes.
In Aim 2 we performed studies of microbial colonization in monocot and dicot roots and leaves. Transcriptional profiling of pathogen and plant highlight common and contrasting principles and forms the basis for future research. We have challenged our findings by testing beneficial fungi to assess commonalities and differences between mutualist and pathogen colonization. We used genetic, cell biology and genomics to find suitable resistance alleles highly relevant to crop production and global food security. At the completion of the project, we had identified SCAR/WAVE genes as suitable candidate genes for plant disease resistance breeding against root oomycete infections.
We cannot solely rely on classical resistance genes to keep ahead of the pathogens. There is an urgent need to develop approaches based on knowledge of the pathogen’s Achilles heel: core plant processes that are required for pathogen colonization.
We reasoned that disease resistance based on manipulation of host accessibility processes has a higher probability for durability, and is best identified using a broad host-range pathogen. Thus, we employed the filamentous pathogen Phytophthora palmivora to mine plant alleles and unravel host processes providing microbial access in roots and leaves of monocot and dicot plants.
In Aim 1 we have utilized plant symbiosis mutants and allelic variation to elucidate general mechanisms of colonization by filamentous microbes. This enabled us to identify genes which help creating an environment conducive to infection by filamentous pathogens. Removal of these genes renders specific plant tissues more resilient to infection. The identified genes can thus be considered susceptibility genes.
In Aim 2 we performed studies of microbial colonization in monocot and dicot roots and leaves. Transcriptional profiling of pathogen and plant highlight common and contrasting principles and forms the basis for future research. We have challenged our findings by testing beneficial fungi to assess commonalities and differences between mutualist and pathogen colonization. We used genetic, cell biology and genomics to find suitable resistance alleles highly relevant to crop production and global food security. At the completion of the project, we had identified SCAR/WAVE genes as suitable candidate genes for plant disease resistance breeding against root oomycete infections.
Aim 1. Utilizing existing plant mutants and allelic variation to elucidate general microbe accessibility mechanisms.
We have identified new genetic components underlying root interactions in Medicago and root and leaf interactions in barley. We assessed mutants impaired in specific symbiotic interactions for their impact on the interaction with an aggressive broad range pathogen. We cloned the gene underlying the M. truncatula api mutation. Our work suggests that specific changes in the plant cell wall at root tips have a dramatic effect on plant microbe interactions without major impacts on overall development. This change in cell wall properties contributes to the quantitative resistance phenotype. These and other findings are published in Gavrin et al., 2020, Current Biology.
We also have utilized natural variation in M. truncatula to identify a GRAS transcription factor as being involved in pathogen and symbiont interaction (Rey et al., 2017, J. Exp. Bot.).
We have shown that barley mlo mutants are more resistant to P. palmivora and that this effect is limited to meristem-proximal leaf tissues (Le Fevre et al, 2016, MPMI). We have used natural variation in economically relevant cereals to map a quantitative trait locus for leaf resistance in a cross between two barley cultivars. This locus coincides with a segregation distortion locus resulting in skewed F2 populations.
Aim 2. Comparative colonisation process studies in different organs of dicot vs. monocot plants
We have established infection systems for roots and leaves of Medicago, Nicotiana benthamiana, barley and wheat including a range of Phytophthora palmivora isolates with varying aggressiveness. We microscopically assessed infection timing in roots and leaves of N. benthamiana and barley using fluorescently labelled P. palmivora. We carried out dual-RNAseq of infected N. benthamiana roots and barley roots and leaves. We have identified a full set of candidate Phytophthora effectors and their conservation in diverse P. palmivora strains. This is a great research for effector-aided resistance breeding.
Most recently we have identified three SCAR/WAVE genes in barley and delineated their phylogeny. Through collaboration with the team of Matthew Moscou (TSL Norwich) we have generated Cas9-free mutants in each individual gene using CRISPR/Cas9 genome editing. These lines form a foundation for future pathogen, symbiont and development studies to test the feasibility of SCAR/WAVE mutants in quantitative resistance strategies.
We have generated tools (SecretSanta, AMfinder) which will help the transcriptome analyses as well as the semiautomated quantification of fungal colonisation processes in roots. We have provided material and pathogen sequence information to others and have disseminated our findings and approaches on conferences, in seminar presentations, via social media as well as in outreach activities. The team has acquired new skills, network connections and several members were able to move into academic and non-academic positions.
We have identified new genetic components underlying root interactions in Medicago and root and leaf interactions in barley. We assessed mutants impaired in specific symbiotic interactions for their impact on the interaction with an aggressive broad range pathogen. We cloned the gene underlying the M. truncatula api mutation. Our work suggests that specific changes in the plant cell wall at root tips have a dramatic effect on plant microbe interactions without major impacts on overall development. This change in cell wall properties contributes to the quantitative resistance phenotype. These and other findings are published in Gavrin et al., 2020, Current Biology.
We also have utilized natural variation in M. truncatula to identify a GRAS transcription factor as being involved in pathogen and symbiont interaction (Rey et al., 2017, J. Exp. Bot.).
We have shown that barley mlo mutants are more resistant to P. palmivora and that this effect is limited to meristem-proximal leaf tissues (Le Fevre et al, 2016, MPMI). We have used natural variation in economically relevant cereals to map a quantitative trait locus for leaf resistance in a cross between two barley cultivars. This locus coincides with a segregation distortion locus resulting in skewed F2 populations.
Aim 2. Comparative colonisation process studies in different organs of dicot vs. monocot plants
We have established infection systems for roots and leaves of Medicago, Nicotiana benthamiana, barley and wheat including a range of Phytophthora palmivora isolates with varying aggressiveness. We microscopically assessed infection timing in roots and leaves of N. benthamiana and barley using fluorescently labelled P. palmivora. We carried out dual-RNAseq of infected N. benthamiana roots and barley roots and leaves. We have identified a full set of candidate Phytophthora effectors and their conservation in diverse P. palmivora strains. This is a great research for effector-aided resistance breeding.
Most recently we have identified three SCAR/WAVE genes in barley and delineated their phylogeny. Through collaboration with the team of Matthew Moscou (TSL Norwich) we have generated Cas9-free mutants in each individual gene using CRISPR/Cas9 genome editing. These lines form a foundation for future pathogen, symbiont and development studies to test the feasibility of SCAR/WAVE mutants in quantitative resistance strategies.
We have generated tools (SecretSanta, AMfinder) which will help the transcriptome analyses as well as the semiautomated quantification of fungal colonisation processes in roots. We have provided material and pathogen sequence information to others and have disseminated our findings and approaches on conferences, in seminar presentations, via social media as well as in outreach activities. The team has acquired new skills, network connections and several members were able to move into academic and non-academic positions.
Our findings are highly relevant to crop production and thus food security. Food security remains one of the most important challenges of our future. Plant breeding, combined with biotechnology, plays a major role in agricultural improvement.
Traditional R gene mediated resistance mechanisms are limited in durability as they often target specific isolates and can be quickly overcome by the pathogens through mutation. Implementing genetic loci that confer a higher degree of resistance into crops is an alternative method to achieve durable resistance. Identifying these loci is the aim of our work. Filamentous pathogens and specifically the genus Phytophthora encompass several of the most notorious plant pathogens affecting worldwide agriculture.
The identified target genes for resistance against P. palmivora (api, mlo, SCAR/WAVE genes of barley) may impact on resistance breeding and biotechnological engineering of food and fibre producing crops. Given the similarity of infection modes to other notorious filamentous pathogens and the fact that the API protein represents a component of a core plant process impacting on symbiotic bacteria colonisation as well as filamentous pathogens, it is likely that our findings will also have impact on generating durable resistance against other crop pathogens with biotrophic lifestyles.
We have accumulated data on the process of tissue colonisation by biotrophic pathogens which will allow us to assess the overlap of root infection with colonisation strategies of mycorrhiza fungi, a field of research that has been neglected despite its importance. Combined with the body of data available for beneficial symbiosis, researchers worldwide will be able to address the specificity and impact of their own findings and select for suitable resistance targets affecting filamentous microbes in general.
Most recently we have used chimeric DNA constructs to delineate the basis for functional specificity of SCAR/WAVE genes. An understanding of the mechanisms underpinning SCAR/WAVE function and specificity broadens our knowledge of this core protein family involved in actin dynamics and may inform gene targeting approaches aimed at inactivating crop SCAR/WAVE genes to achieve quantitative resistance.
Traditional R gene mediated resistance mechanisms are limited in durability as they often target specific isolates and can be quickly overcome by the pathogens through mutation. Implementing genetic loci that confer a higher degree of resistance into crops is an alternative method to achieve durable resistance. Identifying these loci is the aim of our work. Filamentous pathogens and specifically the genus Phytophthora encompass several of the most notorious plant pathogens affecting worldwide agriculture.
The identified target genes for resistance against P. palmivora (api, mlo, SCAR/WAVE genes of barley) may impact on resistance breeding and biotechnological engineering of food and fibre producing crops. Given the similarity of infection modes to other notorious filamentous pathogens and the fact that the API protein represents a component of a core plant process impacting on symbiotic bacteria colonisation as well as filamentous pathogens, it is likely that our findings will also have impact on generating durable resistance against other crop pathogens with biotrophic lifestyles.
We have accumulated data on the process of tissue colonisation by biotrophic pathogens which will allow us to assess the overlap of root infection with colonisation strategies of mycorrhiza fungi, a field of research that has been neglected despite its importance. Combined with the body of data available for beneficial symbiosis, researchers worldwide will be able to address the specificity and impact of their own findings and select for suitable resistance targets affecting filamentous microbes in general.
Most recently we have used chimeric DNA constructs to delineate the basis for functional specificity of SCAR/WAVE genes. An understanding of the mechanisms underpinning SCAR/WAVE function and specificity broadens our knowledge of this core protein family involved in actin dynamics and may inform gene targeting approaches aimed at inactivating crop SCAR/WAVE genes to achieve quantitative resistance.