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Mechanisms of natural auto immunity triggered by plant NLR immune receptors

Periodic Reporting for period 1 - NLRs (Mechanisms of natural auto immunity triggered by plant NLR immune receptors)

Reporting period: 2015-09-01 to 2017-08-31

"How ""phenotypes"" are controlled by ""genotypes"" is the essential question in biology. Understanding natural ""genotype"" or ""genes"" and the mechanism mediated by the ""genotype"" or ""gene"" behind a ""phenotype"" facilitates a more feasible application of science in practical purpose and very important for society and Agriculture. To complement the rapid development of sequencing technique, which has been successfully used to predict potential ""genotypes"" for some agricultural ""phenotypes"" from natural species, functional validation of these predicted ""genotypes"", by interrupting the genes with T-DNA insertion or RNA interference, was traditionally used. However, these methods involve foreign DNA from bacterial in the genome to cause or maintain the interrupting effect, which needs to be GMO plants (defined as containing the foreign DNAs in an organism). On the other hand, the insertion of foreign genes in the genome could cause unexpected consequences such as an opposite effect (enhancing instead of knocking out the function of gene for study depending on the position of T-DNA insertion). CRISPR/Cas9 system has been extensively used as a genome editing tool recently, in which the endonuclease activity of Cas9 digest the specific target DNA sequence under the leading of CRISPR guide RNA sequence. One important feature of this method is that the foreign transgene is able to be excluded from the organism once the mutations are generated, which is not GMOs. So, one of the foundation objectives of this project is to develop an efficient CRISPR/Cas system for knocking out genes from natural species of Arabidopsis, which is an extensively used model plant and has made great contributions to even crop science. Based on this aim in a starting stage, an easy-to-use CRISPR vector toolbox has been developed from this project, with a design convenient to remove the foreign transgenes (non GMO plants). And with this toolbox, both single site mutation and fragment deletion in a given gene can be made.

With this methodology, the second issue to be addressed can be done, which is to understand the evolutionary function of immune genes (NBLRR resistant genes) in plants. Immune response mediated by Resistance (R) protein, with nucleotide binding/leucine rich repeat (NB-LRR or NLR) domains, is one of the most important strategies developed by plants. It is well known that NLRs is the most variable gene family and compose of comparable ratio of plant genome. The feature of extensive sequence diversity and presence/absence polymorphism among populations (presence in some accessions but absence from other accessions) indicates balancing selection and/or rapid diversification happened to this gene family during evolution. The objective for this part of project is to understand whether the relatively conserved NLR genes among 1001 Arabidopsis accessions have the same fitness contributions or the balanced fitness contributions to the ones with presence/absence polymorphism. This study will be important for the society because, on one hand, it will help to have a better understanding on the further interaction between pathogen and plants in a much longer time window, and on the other hand, the discovery of important NLR genes that are retained after long-term evolution and contribute to plant fitness will help plant breeders to improve plant performance.
1) Analyzed the 1001 Arabidopsis genomes short read sequencing data using Col-0 as a reference genome, and obtained a list of 32 conserved NLR genes (out of 163 NLR genes for analysis), in collaboration with PhD student Darya Karelina.
2) Set up an easy-to-use CRISPR vector toolbox that is also very efficient, both in generating Indels (Insertion/deletions) or big fragment deletions and in excluding the transgenes from the mutant organisms.
3) To fast obtain plants with specific mutations or deletions, effort has also been put to test a mesophyll protoplast regeneration system. Regeneration was not working well from Arabidopsis protoplast but protoplast transient transfection system is beneficial for this project because it can be used to fast test the efficiency of a pair of sgRNAs for deletion. CRISPR KO multiple genes together was also tried, but it was not working.
4) Using the vector tools and system developed above, knocking out of 53 NLR genes including the 32 genes in the above list and some published functional NLR genes was conducted in Arabidopsis reference genome, and transgene free homozygous or heterozygous plants for 25 out of that have been obtained.
5) The available NLR homozygous or heterozygous mutants have been used for phenotyping screening, which is currently ongoing. Growth rate of seedlings under 23 and 16 degrees were monitored with a RAPA (Raspberry Pi Automated Phenotyping Array) system. Also, the fitness of these mutants are measured currently compared with wild type. In addition, the immunity phenotypes of these mutants together with wild type control are being tested as well, such as the SA level under normal and pathogen infection conditions, and the bacterial growth in the infected leaves. Response to pathogen such as bacterial (Pseudomonas syringae) and downy mildew pathogens (Hpa) are also exhaustively being tested.
6) Subcellular localization of RPW8 proteins from different accessions (KZ10 and Ms-0) has been tested with Confocal. The allele that can cause cell death in hybrid necrosis largely localized in stomatal cells in mesophyll as well as in root cortex. This is the first finding and will be involved in a bigger project regarding RPW8 and RPP7 interaction in the group.
During the supported MC project, two collaborative publications have been published as open access, using my expertise of CRISPR and Microscopy (attached). More major scientific publications are expected to come out mainly from the supported project. And the topics will cover from CRISPR technology (in submission), evolutionary functions of NLR genes (in preparation), NLR genes' function in shaping microbiome (in preparation), and downstream signaling study of NLR genes. Two more collaborative manuscripts are in preparation regarding RPW8 and RPP7. Importantly, the materials generated will be resource for my future own lab.
It will also have great impact from a broader perspective. The potential results from this project, studying plant pathogen interaction in the angle of evolution, will bring implications for plant science communities including breeders in industry and also plant scientists in academia, and colleagues from both areas will do their best to serve for the public better, at least in terms of improve crop qualities. In addition, one effort from this project is put to solve the debatable issue of GMOs (genetically modified organisms) by engineering gene of interest without the retaining of transgenes from other organisms, which is a breakthrough for food security. This will be a good example to be introduced to the public such as school students to get rid of the unnecessary panic towards GMOs.
joint publication 2
joint publication 1