Periodic Reporting for period 4 - RINFEC (The Roots of Infection)
Periodo di rendicontazione: 2024-04-01 al 2024-09-30
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.
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.
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.
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.
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. 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.
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. A procedure for protoplasting root cell was established and optimized.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. To optimize characterization of regulator genes identified in the single cell approach an effort to optimize the procedure for stable transformation and for generating CRISPR mutants was initiated. Stable transformed Lotus lines expressing TurBoID tagged receptors has been established and the first step towards biochemical identification of important protein-protein interactions taken.
To expand identification of genes controlling intercellular infection an EMS mutagenesis and mutant screening program was initiated on Lotus burttii. More than 5000 M1 plants were propagated in greenhouses and seeds were harvested from individual plants and in bulk. The M2 is screened for root nodule phenotypes after inoculation with Sinorhizobium fredii that infects Lotus burttii intercellularly. This unbiased screen has the potential to uncover a new layer of genes and mechanisms regulating rhizobial infection and endophyte colonization will also be monitored in plant mutants identified to assess the possible conservation of intercellular infection mechanisms. Single cell sequencing using the 10X system will further support this approach.
Research on intercellular infection initiated in RINFEC is continued in other projects in order to complete the many promising lines of investigation. The approaches and results obtained in RINFEC has established a foundation for scientific progress in understanding plant microbe-interaction and will have an impact on application of biologicals that is an emerging industry. RINFEC research was dissiminated at international and national conferences and through national and international media.
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. A procedure for protoplasting root cell was established and optimized.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. To optimize characterization of regulator genes identified in the single cell approach an effort to optimize the procedure for stable transformation and for generating CRISPR mutants was initiated. Stable transformed Lotus lines expressing TurBoID tagged receptors has been established and the first step towards biochemical identification of important protein-protein interactions taken.
To expand identification of genes controlling intercellular infection an EMS mutagenesis and mutant screening program was initiated on Lotus burttii. More than 5000 M1 plants were propagated in greenhouses and seeds were harvested from individual plants and in bulk. The M2 is screened for root nodule phenotypes after inoculation with Sinorhizobium fredii that infects Lotus burttii intercellularly. This unbiased screen has the potential to uncover a new layer of genes and mechanisms regulating rhizobial infection and endophyte colonization will also be monitored in plant mutants identified to assess the possible conservation of intercellular infection mechanisms. Single cell sequencing using the 10X system will further support this approach.
Research on intercellular infection initiated in RINFEC is continued in other projects in order to complete the many promising lines of investigation. The approaches and results obtained in RINFEC has established a foundation for scientific progress in understanding plant microbe-interaction and will have an impact on application of biologicals that is an emerging industry. RINFEC research was dissiminated at international and national conferences and through national and international media.
We expect to profile single cells of Lotus japonicus inoculated with rhizobial bacteria that use different infection modes by the 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. Expanding this investigation beyond species boundaries an unbiased genetic approach on intercellular infection of Lotus burttii by Sinorhizobium fredii combine d with single cell sequencing is in progressing.
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. This will increase our understanding of plant interaction with microbiota in the rhizosphere and endosphere and provide a deeper understanding of rhizobial infection mechanism found among the many plant species in the legume family.
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. This will increase our understanding of plant interaction with microbiota in the rhizosphere and endosphere and provide a deeper understanding of rhizobial infection mechanism found among the many plant species in the legume family.