Division of Biochemistry, University of Missouri, Columbia, MO, 65211, USA.
Division of Biological Sciences, University of Missouri, Columbia, MO, 65211, USA.
J Integr Plant Biol. 2022 Aug;64(8):1531-1542. doi: 10.1111/jipb.13309. Epub 2022 Jul 18.
Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4 (MKK4) and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YODA (YDA), also known as MAPK KINASE KINASE4 (MAPKKK4), is upstream of MKK4/MKK5 and forms a complete MAPK cascade (YDA-MKK4/MKK5-MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5-MPK3/MPK6 module. However, the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 pathogen-associated molecular pattern (PAMP) treatment suggests the presence of additional MAPKKK(s) in this MAPK cascade in signaling plant immunity. To investigate whether YDA is also involved in plant immunity, we attempted to generate mapkkk3 mapkkk5 yda triple mutants. However, it was not possible to recover one of the double mutant combinations (mapkkk5 yda) or the triple mutant (mapkkk3 mapkkk5 yda) due to a failure of embryogenesis. Using the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9) approach, we generated weak, N-terminal deletion alleles of YDA, yda-del, in a mapkkk3 mapkkk5 background. PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant, and the triple mutant was more susceptible to pathogen infection, suggesting YDA also plays an important role in plant immune signaling. In addition, MAPKKK5 and, to a lesser extent, MAPKKK3 were found to contribute to gamete function and embryogenesis, together with YDA. While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant, mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal, similar to the mpk3 mpk6 double mutants. These results demonstrate that YDA, MAPKKK3, and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5-MPK3/MPK6 module in both plant immunity and growth/development.
拟南芥丝裂原活化蛋白激酶 3（MAPK3 或 MPK3）和 MPK6 在植物免疫和生长/发育中发挥重要的信号作用。在这些过程中，MAPK 激酶 4（MKK4）和 MKK5 在 MPK3 和 MPK6 的上游发挥冗余作用。YODA（YDA），也称为 MAPK 激酶激酶 4（MAPKKK4），位于 MKK4/MKK5 的上游，并在调节植物生长和发育中形成一个完整的 MAPK 级联（YDA-MKK4/MKK5-MPK3/MPK6）。在植物免疫中，MAPKKK3 和 MAPKKK5 在相同的 MKK4/MKK5-MPK3/MPK6 模块的上游发挥冗余作用。然而，在 flg22 病原体相关分子模式（PAMP）处理后，mapkkk3 mapkkk5 双突变体中 MPK3/MPK6 的残留激活表明，在这个 MAPK 级联中存在额外的 MAPKKK（s），用于信号转导植物免疫。为了研究 YDA 是否也参与植物免疫，我们试图生成 mapkkk3 mapkkk5 yda 三突变体。然而，由于胚胎发生失败，无法恢复其中一种双突变体组合（mapkkk5 yda）或三突变体（mapkkk3 mapkkk5 yda）。使用成簇规律间隔短回文重复（CRISPR）-CRISPR 相关蛋白 9（Cas9）方法，我们在 mapkkk3 mapkkk5 背景下生成了 YDA 的弱、N 端缺失等位基因 yda-del。在 mapkkk3 mapkkk5 yda-del 突变体中，PAMP 触发的 MPK3/MPK6 激活进一步降低，三突变体对病原体感染更敏感，表明 YDA 也在植物免疫信号转导中发挥重要作用。此外，发现 MAPKKK5 以及在较小程度上，MAPKKK3 与 YDA 一起，共同参与配子功能和胚胎发生。虽然 mapkkk3 yda 双纯合突变体表现出与 yda 单突变体相同的生长和发育缺陷，但 mapkkk5 yda 双突变体和 mapkkk3 mapkkk5 yda 三突变体胚胎致死，类似于 mpk3 mpk6 双突变体。这些结果表明，YDA、MAPKKK3 和 MAPKKK5 在 MKK4/MKK5-MPK3/MPK6 模块的上游具有重叠功能，在植物免疫和生长/发育中均有作用。
