A genetic screen to identify factors that regulate meiotic recombination in Arabidopsis
DR. DIVYASHREE NAGESWARAN
This is a continuation of the article on “Kinases & Phosphatases II” from the January 21 edition. The former article described various known protein phosphatases, their structure, and key functions exposed in model systems. Here in this article, I’ve written about meiosis & recombination, and how we arrived at identifying Protein Phosphatase X1/PP4 using a genetic screen in Arabidopsis thaliana. However, the conclusions we derived from experiments that supported the role of PPX1/PP4 in meiotic recombination will be discussed in the next issue. Importantly, the above title formed my Ph.D. dissertation (https://doi.org/10.17863/CAM.35722) under the supervision of Prof. Ian Henderson (University of Cambridge, UK) and Dr. Kyuha Choi (Pohang University, S.Korea). Also, the piece of work has been recently published in “Nature Plants” under the title, “HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis” (https://doi.org/10.1038/s41477-021-00889-y). Please subscribe to the article for full access.
Meiosis, also called “reductional division”, is a specialized form of eukaryotic cell division, which involves a single round of DNA replication followed by two rounds of chromosome segregation, producing haploid cells that fuse to form a diploid cell upon fertilization. During meiotic prophase I stage pachytene, homologous chromosomes pair and undergo reciprocal exchange of DNA from programmed double-stranded breaks (DSBs), producing crossovers or non-crossovers (gene conversions) between chromosomes. This mechanism of reciprocal exchange is called “recombination” and the resultant products are termed as crossovers (CO). Meiotic crossovers, that result from proper chromosome segregation and restoration of ploidy level, promote genetic diversity that can enhance the efficiency of natural selection and adaptation. Wide genetic diversity is a great asset and resource in plant breeding programs for crop improvement.
Despite more DSBs in eukaryotes, CO is restricted in number and distribution. According to numbers, of the 250 DSBs generated, only 10 COs (5 Chr pairs) are formed per meiotic cell in Arabidopsis ecotypes or natural accessions. With the help of light microscopy, we could visualize all the meiosis stages, including the most happening prophase I (where DSBs and COs occur), for which we prepare DAPI-stained (a DNA dye) chromosome spreads using pollen mother cells (PMCs). Cytology works shown in the published article, and their implications will be discussed in the next issue.
The recombination machinery that is processed in a meiocyte is governed by a set of key proteins. The majority of plant COs, about 85% are dependent on Class I CO interfering repair, and only a few are formed via the Class II non-interfering pathway. Not all proteins involved in the recombination pathway across eukaryotes are conserved for their functions unlike the DSB protein, SPO11. Moreover, Class II CO repair is restricted by anti-crossover pathways, however, there isn’t anything similar that is identified to repress the Class I CO pathway. This enhanced our scope to find new factors or genes that could control recombination. Therefore, we carried out a forward genetic screen via ethyl methanesulfonate (EMS) mutagenesis in Arabidopsis. Using fluorescent crossover reporters, we screened for mutants with higher or lower crossover rates (centiMorgan, cM) from a large mutant population. These mutants were compared with wild type (parent) crossover frequency (~ 20 cM). Of the several mutants we identified, only five were confirmed heritable. Again, of the five heritable mutants, two were found to be the alleles of known recombination factors (Figure 1). Further, we chose to focus on the high crossover rate 1 (hcr1) mutant, developed a mutant backcross F2 (BC1F2) population, and performed mapping-by-deep sequencing (NGS) to identify the candidate mutation site (Figure 1). Using SHOREMAP, we found hcr1 mutation in the gene At4g26720 (located in Chr 4) that encodes PROTEIN PHOSPHATASE X1 (PPX1), a catalytic subunit of the nuclear Serine/Threonine (S/T) protein phosphatase PP4 complex (Figure 2). After candidate gene identification, we performed a genetic complementation test using genetic analysis (cross a T-DNA insertion variant with EMS mutant and measure recombination in F1 hybrids) and transgenic (introducing a whole genomic fragment of PPX1 into the mutant hcr1 background using Agrobacterium-mediated transformation) methods. This validated the gene PPX1 as HCR1 (Figure 2).
Readers, you will find more depth about the genetic screen pipeline, fluorescent crossover reporter system, isolated mutants, SHOREMAP showing the hcr1 mutation site in Chr 4, gene structure of PPX1, and validation tests (as in Figures 1-2) from the published version of the article (https://doi.org/10.1038/s41477-021-00889-y). The next issue will encompass the remaining figures portraying the experimental data that propose the role of PPX1/PP4 in the context of recombination.
Nageswaran DC, Kim J, Lambing C, Kim J, Park J, Kim EJ, Cho HS, Kim H, Byun D, Park YM, Kuo P, Lee S, Tock AJ, Zhao X, Hwang I, Choi K, Henderson IR. HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis. Nat Plants. 2021 Apr;7(4):452-467. DOI: 10.1038/s41477-021-00889-y.