Conditional genetic screen in <i>Physcomitrella patens</i> reveals a novel microtubule depolymerizing-end-tracking protein

Our ability to identify genes that participate in cell growth and division is limited because their loss often leads to lethality. A solution to this is to isolate conditional mutants where the phenotype is visible under restrictive conditions. Here, we capitalize on the haploid growth-phase of the moss Physcomitrella patens to identify conditional loss-of-growth (CLoG) mutants with impaired growth at high temperature. We used whole-genome sequencing of pooled segregants to pinpoint the lesion of one of these mutants (clog1) and validated the identified mutation by rescuing the conditional phenotype by homologous recombination. We found that CLoG1 is a novel and ancient gene conserved in plants. At the restrictive temperature, clog1 plants have smaller cells but can complete cell division, indicating an important role of CLoG1 in cell growth, but not an essential role in cell division. Fluorescent protein fusions of CLoG1 indicate it is localized to microtubules with a bias towards depolymerizing microtubule ends. Silencing CLoG1 decreases microtubule dynamics, suggesting that CLoG1 plays a critical role in regulating microtubule dynamics. By discovering a novel gene critical for plant growth, our work demonstrates that P. patens is an excellent genetic system to study genes with a fundamental role in plant cell growth.由于参与细胞生长与分裂的基因一旦缺失往往会导致个体死亡，因此我们对这类基因的鉴定能力受到极大限制。针对该问题，可通过分离在限制性培养条件下可呈现目标表型的条件突变体加以解决。本研究利用小立碗藓（Physcomitrella patens）的单倍体生长阶段，筛选得到在高温条件下生长受损的条件性生长缺失（conditional loss-of-growth, CLoG）突变体。我们通过混合分离体全基因组测序，精准定位到其中一株突变体（clog1）的致病变异位点，并通过同源重组拯救该条件表型，验证了所鉴定的突变。我们发现CLoG1是一类在植物中保守存在的新型古老基因。在限制性高温条件下，clog1突变体植株的细胞体积更小，但仍可完成细胞分裂，这表明CLoG1在细胞生长过程中发挥重要作用，但并非细胞分裂所必需。CLoG1的荧光蛋白融合标记实验显示，该蛋白定位于微管，且偏好富集于微管解聚末端。沉默CLoG1会降低微管动态性，这提示CLoG1在调控微管动态性过程中发挥关键作用。本研究通过发现一类对植物生长至关重要的新型基因，证明了小立碗藓（P. patens）是研究植物细胞生长核心功能基因的绝佳遗传体系。