Periodic Reporting for period 2 - RINFEC (The Roots of Infection)
Période du rapport: 2021-04-01 au 2022-09-30
What is the problem/issue being addressed?
Plant roots and soil microbes have been associated since plants colonized the earth. Nevertheless the mechanisms that have evolved to control the interaction with the very diverse population of soil microbes are currently unknown. RINFEC will identify both plant and bacterial genes involved in root colonization by bacterial microbiota especially endophytes. The importance of the plant root microbiota for plant development and plant health is a new hitherto overlooked research field and only now beginning to emerge. Research on plants and their naturally associated microorganisms is therefore in a prime position to provide new perspectives and concepts for understanding plant function, performance and plant growth under limited input conditions. This will provide opportunities to reduce the environmental footprint and could also define breeding targets and develop applications through microbial interventions.
Why is it important for society?
One of the grand challenges facing humanity is to secure sufficient and healthy food for the increasing world population. This involves sustainable cultivation of crop plants under changing climate conditions. The aim and perspective of RINFEC is to provide knowledge and tools for evidence-based development of new resilient crops and associated microbial interventions that will improve productivity, reduce the need for fertilizers and pesticides, and alleviate negative environmental impact accompanying our food production.
What are the overall objectives?
One of the limitations in microbiome research is the lack of genetically tractable and robust experimental systems suitable for investigating bacterial endophyte infection of the root interior and processes that restrict root the surface colonizers in the rhizosphere/rhizoplane from progressing to the plant interior. RINFEC´s central hypothesis is that key components of an ancient pathways for bacterial colonization of the root surface (rhizosphere) and root interior (endosphere) were adapted during evolution of mechanism(s) controlling colonization of legume roots by symbiotic rhizobia. RINFEC will uncover the genetics and biochemistry of these shared mechanisms by characterizing a novel, unexplored intercellular infection mode observed for certain rhizobia like the IRBG74 strain that act as endophytes in non-legume plants and are able to infect and induce nitrogen fixing root nodule in the model legume Lotus japonicus and related species.
Plant roots and soil microbes have been associated since plants colonized the earth. Nevertheless the mechanisms that have evolved to control the interaction with the very diverse population of soil microbes are currently unknown. RINFEC will identify both plant and bacterial genes involved in root colonization by bacterial microbiota especially endophytes. The importance of the plant root microbiota for plant development and plant health is a new hitherto overlooked research field and only now beginning to emerge. Research on plants and their naturally associated microorganisms is therefore in a prime position to provide new perspectives and concepts for understanding plant function, performance and plant growth under limited input conditions. This will provide opportunities to reduce the environmental footprint and could also define breeding targets and develop applications through microbial interventions.
Why is it important for society?
One of the grand challenges facing humanity is to secure sufficient and healthy food for the increasing world population. This involves sustainable cultivation of crop plants under changing climate conditions. The aim and perspective of RINFEC is to provide knowledge and tools for evidence-based development of new resilient crops and associated microbial interventions that will improve productivity, reduce the need for fertilizers and pesticides, and alleviate negative environmental impact accompanying our food production.
What are the overall objectives?
One of the limitations in microbiome research is the lack of genetically tractable and robust experimental systems suitable for investigating bacterial endophyte infection of the root interior and processes that restrict root the surface colonizers in the rhizosphere/rhizoplane from progressing to the plant interior. RINFEC´s central hypothesis is that key components of an ancient pathways for bacterial colonization of the root surface (rhizosphere) and root interior (endosphere) were adapted during evolution of mechanism(s) controlling colonization of legume roots by symbiotic rhizobia. RINFEC will uncover the genetics and biochemistry of these shared mechanisms by characterizing a novel, unexplored intercellular infection mode observed for certain rhizobia like the IRBG74 strain that act as endophytes in non-legume plants and are able to infect and induce nitrogen fixing root nodule in the model legume Lotus japonicus and related species.
The Lotus japonicus-IRBG74 symbiosis was established as a novel working model to study the conditional intercellular infection in legumes and the main features of the Lotus-IRBG74 symbiosis was characterized. RNAseq analysis and the symbiotic performance of Lotus mutants allowed the elucidation of the transcriptome and genetic requirements for intercellular colonization in this symbiotic association. A transcriptome analysis has provided a list of Lotus genes mainly induced by IRBG74. The list was assembled by comparing the transcriptomic response of Lotus roots during an intra- and intercellular infection process. Homozygous Lotus LORE1 mutants were obtained for some of these genes and their symbiotic performance assessed. The characterization of two mutants with a clear symbiotic perturbance along with a root/root hair phenotype, represents the achievement of this approach and additional mutant characterization is in progress.
Complementing these approaches a Lotus LORE1 mutant population was screened for mutants impaired in intercellular infection. Screening a population of > 200,000 mutants induced by the LORE1 retrotransposon allowed isolation of > 100 mutants with a symbiotic phenotype. A valuable biological resource for future research. Additionally, a list of genes potentially responsible for the deficient symbiotic ability in this mutant population was obtained. Besides the symbiotic phenotype, at least one selected mutant exhibits an interesting and unexpected root phenotype, whose characterization would impact other plant science fields.
To explore the biodiversity among Lotus japonicus ecotypes a GWAS approach was performed using more than 100 Lotus ecotypes from which genome sequences was available. Genomic regions associated to intercellular infection ability were identified by analyzing the nodulation performance with IRBG74 in > 100 Lotus japonicus ecotypes. Currently, we are evaluating the symbiotic capacity of mutants affected in genes associated to these regions and the role played during intercellular infection mechanism.
Taking a completely different approach to identify Lotus genes required for or involved in governing the rhizobial infection, a single cell sequencing project was initiated. First a procedure for protoplasting root cell was established and optimized. Protoplast was then cleaned up by centrifugation debris away before single cell sequencing was performed using the 10X methodology. Both wild-type plants and mutants known to be impaired in infection thread formation like cyclops were subjected to protoplast isolation and single cell sequencing. in order to identify novel regulators of infection. This has yielded an exceptionally detailed map of the infection process in different cell types of the root and root nodules. In order to optimize the characterization of regulator genes identified in the single cell approach an effort to optimize the procedure for stable transformation of Lotus and for generating CRISPR mutants was initiated. Using this optimized procedure stable transformed Lotus lines expressing TurBoID tagged receptors has been established and the first step towards biochemical investigation of the infection processes and identification of important protein-protein interactions has been taken.
Complementing these approaches a Lotus LORE1 mutant population was screened for mutants impaired in intercellular infection. Screening a population of > 200,000 mutants induced by the LORE1 retrotransposon allowed isolation of > 100 mutants with a symbiotic phenotype. A valuable biological resource for future research. Additionally, a list of genes potentially responsible for the deficient symbiotic ability in this mutant population was obtained. Besides the symbiotic phenotype, at least one selected mutant exhibits an interesting and unexpected root phenotype, whose characterization would impact other plant science fields.
To explore the biodiversity among Lotus japonicus ecotypes a GWAS approach was performed using more than 100 Lotus ecotypes from which genome sequences was available. Genomic regions associated to intercellular infection ability were identified by analyzing the nodulation performance with IRBG74 in > 100 Lotus japonicus ecotypes. Currently, we are evaluating the symbiotic capacity of mutants affected in genes associated to these regions and the role played during intercellular infection mechanism.
Taking a completely different approach to identify Lotus genes required for or involved in governing the rhizobial infection, a single cell sequencing project was initiated. First a procedure for protoplasting root cell was established and optimized. Protoplast was then cleaned up by centrifugation debris away before single cell sequencing was performed using the 10X methodology. Both wild-type plants and mutants known to be impaired in infection thread formation like cyclops were subjected to protoplast isolation and single cell sequencing. in order to identify novel regulators of infection. This has yielded an exceptionally detailed map of the infection process in different cell types of the root and root nodules. In order to optimize the characterization of regulator genes identified in the single cell approach an effort to optimize the procedure for stable transformation of Lotus and for generating CRISPR mutants was initiated. Using this optimized procedure stable transformed Lotus lines expressing TurBoID tagged receptors has been established and the first step towards biochemical investigation of the infection processes and identification of important protein-protein interactions has been taken.
We expect to profile single cells of Lotus inoculated with rhizobial bacteria that use different infection by end of project. IRBG74 is used for characterizing the intercellular infection process while a standard M. loti R7A strain is used for the comparative intracellular infection process. Taking this comparative approach and spatial transcriptomics on longitudinal/cross sections a confidently call of all gene expression clusters is expected at the end of project .
Altogether this will enable identification of novel regulators in intercellular and intracellular infections based on infected cell clusters. Positive and negative regulators of intercellular and intracellular infection can subsequently be filtered by combining single cell RNAseq and bulk RNAseq
Altogether this will enable identification of novel regulators in intercellular and intracellular infections based on infected cell clusters. Positive and negative regulators of intercellular and intracellular infection can subsequently be filtered by combining single cell RNAseq and bulk RNAseq