Similar to the experiments done by Lebeis et al. [1], first we must define a host and study its immune system. For Arabidopsis thaliana, salicylic acid (SA) is the one of the main compounds in its immune system that helps shape microbiomes recruitment and assembly [1]. The experimental design will to allow us to determine which functions allow microbial colonizers to be robust or sporadic is as follows:
a. Prepare a greenhouse and divide it into nine grid squares. Ensure that the greenhouse has temperature control alongside light control functions. Fix the temperature and light cycles optimal for A. thaliana growth.
b. Obtain A. thaliana wild type seeds, A. thaliana seeds of that overexpress SA (e.g. cpr1) and seeds of mutants that under-express
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1Analyse the community data from 16S rRNA sequencing and metagenomics Identify which colonizers are found with more than 50% probability in wildtype plants (robust colonizers) and those with less than 50% probability in wildtype plants (sporadic colonizers). Identify any overlapping colonizers between wild type and mutants breeds and which colonizers are only present in SA overproducing (if any) or SA deficient mutants (if any).
i. 1Analyse the metagenome and metatranscriptome data and attempt to identify all the genes. Look out for genes that are constitutively expressed in the microbiome community samples from SA overproducing plants that are either at increased levels as compared to wild type or absent in wild type. Link these genes to the microbial members that contain them if possible and see if they correspond to robust colonizers. If so, these genes should be related to functions for microbial robustness.
j. 1Look out for genes from the metagenome and metatranscriptome data that are constitutively expressed in the microbiome community samples from SA deficient plants that are either at increased levels as compared to wild type or absent in wild type. Link these genes to the microbial members that contain them if possible and see if they correspond to sporadic colonizers. If so, these genes should be related to microbial functions that allow for them to be
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1Create transposon libraries for robust or sporadic microbial community members identified in step (h) if possible.
l. 1Add the transposon library mutants (mutants of bacteria that are considered robust) to sterile roots of A. thaliana wild type plants and mutants that overexpress SA and see which library mutants are not able to colonize the plants anymore or poorly colonize SA overproducing plants and perhaps the wild type plants as well. These mutants have lost genes that are important for robustness.
m. 1Add the transposon library mutants (mutants of bacteria that are considered sporadic) to sterile roots of A. thaliana wild type plants and mutants that are SA deficient and see which library mutants are not able to colonize the plants anymore or poorly colonize SA deficient plants. These mutants have lost genes that are important for them to be sporadic.
n. 1Extract the DNA from mutants determined in steps (l) and (m) and sequence them to figure out where the transposons have inserted themselves into the genome and which genes were disrupted. Compare the results with metagenome and metatranscriptome