|
磷脂酰肌醇3-磷酸(PI3P)是自噬和内体分选的重要膜标志，在植物中由Ⅲ类磷脂酰肌醇3-激酶(PI3K)复合物合成，PI3K由Vps34激酶、ATG6、VPS15和VPS38或ATG14作为第四亚单位组成。虽然缺少这三个核心亚单位的拟南芥植株是不育的，但vps38突变体是可行的，但具有异常的叶片、根和种子发育、蔗糖感知和内体运输，这表明VPS38和ATG14是非冗余的。 在这里，我们通过一组CRISPR/Cas9和T-DNA插入突变体扰乱两个拟南芥近缘基因来评估ATG14的作用。Atg14a atg14b双突变体是相对正常的表型，但在营养胁迫期间表现出自噬缺陷，包括自噬小体积累减少和货物运输减少。 出人意料的是，纯合atg14a atg14b vps38三重突变体是可行的，但表现出严重的玫瑰花结发育，并降低了繁殖力、花粉萌发和自噬，这与ATG14和VPS38都需要完全激活PI3P生物学是一致的。然而，三个突变体仍然积累了PI3P，但对PI3K抑制剂wortmannin敏感，表明ATG14/VPS38组分对PI3P的合成不是必需的。 总而言之，ATG14/VPS38突变体为研究在PI3P生物学植物特定功能提供可能。 Phosphatidylinositol 3-phosphate (PI3P) is an essential membrane signature for both autophagy and endosomal sorting that is synthesized in plants by the class-III phosphatidylinositol 3-kinase (PI3K) complex, consisting of the VPS34 kinase, together with ATG6, VPS15, and either VPS38 or ATG14 as the fourth subunit. While Arabidopsis plants missing the three core subunits are infertile, vps38 mutants are viable but have aberrant leaf, root and seed development, sucrose sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are non-redundant. Here, we evaluated the role of ATG14 through a collection of CRISPR/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. atg14a atg14b double mutants were relatively normal phenotypically but displayed autophagy defects, including reduced accumulation of autophagic bodies and cargo delivery during nutrient stress. Unexpectedly, homozygous atg14a atg14b vps38 triple mutants were viable but showed severely compromised rosette development and reduced fecundity, pollen germination and autophagy, consistent with a need for both ATG14 and VPS38 to fully actuate PI3P biology. However, the triple mutants still accumulated PI3P, but were hypersensitive to the PI3K inhibitor wortmannin, indicating that the ATG14/VPS38 component is not essential for PI3P synthesis. Collectively, the ATG14/VPS38 mutant collection now permits the study of plants altered in specific aspects of PI3P biology  |
|
|
