Periodic Reporting for period 3 - ACHILLES-HEEL (Crop resistance improvement by mining natural and induced variation in host accessibility factors)
Reporting period: 2018-09-01 to 2020-02-29
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.
Our hypothesis is 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. We employ 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 utilize plant symbiosis mutants and allelic variation to elucidate general mechanisms of colonization by filamentous microbes. Importantly, we study allelic variation in economically relevant barley to allow immediate translation into breeding programs.
In Aim 2 we perform a comparative study of microbial colonization in monocot and dicot roots and leaves. Transcriptional profiling of pathogen and plant will highlight common and contrasting principles and illustrate the impact of differential plant anatomies. We challenge our findings by testing beneficial fungi and nitrogen fixing symbiosis to assess commonalities and differences between mutualist and pathogen colonization. We use 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 will have a set of candidate genes for plant disease resistance breeding.
We have made significant progress in identifying genetic components underlying root interactions in Medicago and leaf interactions in barley and are continuing to characterise the underlying genes and molecular mechanisms.
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 and have identified that api mutant plants have modified cell wall properties possibly leading to resistance. We have characterised api mutants for their pathogen phenotypes, their nitrogen fixing symbiosis phenotype and the short root hair phenotype. Our work suggests that specific changes in the plant cell wall have a dramatic effect on plant microbe interactions without major impacts on overall development. Our work further indicates that it is the early surface penetration stage where the pathogen fails to enter efficiently (milestone 1).
We have now data showing that the Arabidopsis SCAR2 gene can complement M. truncatula api mutants suggesting functional conservation. Vice versa, Homolog-of-API1 (HAPI1) but not API can complement the Arabidopsis scar2/dis3 mutant distorted leaf trichome phenotype suggesting that Medicago API and its closest homolog HAPI1 fulfil partly different functions. We have furthermore carried out promoter-GUS reporter assays using API, HAPI1 and HAPI2 promoters and found largely constitutive expression of all 3 promoters with a stronger expression in root meristems.
Our transcriptional analysis has identified a xyloglucan transferase/hydrolase whose expression is regulated in an api-dependent manner. We are currently studying its functional relevance for infection.
A role of xyloglucans is evident from our recent finding that api roots secrete much less xyloglucans into their environment. We have demonstrated this through seedling membrane blots probed with xyloglucan specific monoclonal antibodies.
We also have utilized natural variation in M. truncatula to identify a GRAS transcription factor as being involved in pathogen and symbiont interaction (milestone 2).
We have also 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)(milestone 3 partially completed). We have used natural variation in economically relevant cereals to identify and map a quantitative trait locus which renders barley leaves resistant to P. palmivora in a cross between two barley cultivars which respond differently to leaf infection. Interestingly this locus coincides with a segregation distortion locus resulting in skewed F2 populations (towards milestone 4).
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. We microscopically assessed infection timing in roots and leaves of N. benthamiana and barley using fluorescently labelled P. palmivora. We carried out RNAseq of infected N. benthamiana roots and infected barley leaves. We have established a strategy for dual RNAseq where obtained sequence reads are split into plant and microbe by aligning to the references. We confirmed transcriptional changes of selected genes using qRT-PCR. 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.
We have provided material and pathogen sequence information to several researchers including Cenipalma, Colombia, Dr. M. Tian, Hawaii, Prof. S. Kamoun, TSL Norwich and Dr. T. Bozkurt, Imperial College, London.
Traditional R gene mediated dominant 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 including the Irish potato famine causal organism P. infestans.
Our already identified target genes for resistance against P. palmivora (api, mlo) 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 such as rust fungi.
We have accumulated new 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.