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. Although Arabidopsis (Arabidopsis thaliana) plants missing the three core subunits are infertile, vps38 mutants are viable but have aberrant leaf, root, and seed development, Suc sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are nonredundant. Here, we evaluated the role of ATG14 through a collection of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. atg14a atg14b double mutants were relatively normal phenotypically but displayed pronounced 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 they 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.

磷脂酰肌醇3-磷酸酯（PI3P）是自噬和内体分选的必不可少的膜特征，它是由III类磷脂酰肌醇3-激酶（PI3K）复合物在植物中合成的，该复合物由VPS34激酶，ATG6，VPS15和VPS38或ATG14作为第四个子单元。虽然缺少三个核心亚基的拟南芥植物不育，但vps38突变体是可行的，但叶片，根和种子发育异常，蔗糖感测和内体运输异常，表明VPS38和ATG14是非冗余的。在这里，我们通过收集破坏两个拟南芥旁系同源物的CRISPR / Cas9和T-DNA插入突变体评估了ATG14的作用。atg14a atg14b双重突变体在表型上相对正常，但显示出自噬缺陷，包括在营养胁迫期间自噬体的积累减少和货物运输减少。出乎意料的是，纯合的atg14a atg14b vps38三重突变体是可行的，但显示出玫瑰花发育受到严重损害，繁殖力，花粉萌发和自噬减少，这与ATG14和VPS38都需要充分激活PI3P生物学相一致。然而，三重突变体仍积累PI3P，但对PI3K抑制剂渥曼青霉素高度敏感，表明ATG14 / VPS38组分对于PI3P合成不是必需的。总体而言，ATG14 / VPS38突变体集合现在允许研究PI3P生物学特定方面发生变化的植物 花粉萌发和自噬，这与需要ATG14和VPS38充分激活PI3P生物学相一致。然而，三重突变体仍积累PI3P，但对PI3K抑制剂渥曼青霉素高度敏感，表明ATG14 / VPS38组分对于PI3P合成不是必需的。总体而言，ATG14 / VPS38突变体集合现在允许研究PI3P生物学特定方面发生变化的植物 花粉萌发和自噬，这与需要ATG14和VPS38充分激活PI3P生物学相一致。然而，三重突变体仍积累PI3P，但对PI3K抑制剂渥曼青霉素高度敏感，表明ATG14 / VPS38组分对于PI3P合成不是必需的。总体而言，ATG14 / VPS38突变体集合现在允许研究PI3P生物学特定方面发生变化的植物