柱花草SgMPK6互作蛋白的筛选与验证

丝裂原活化蛋白激酶(MAPK)级联反应在调节植物免疫中起关键作用。柱花草炭疽病是危害柱花草生产的严重病害,柱花草SgMPK6基因具有抗炭疽菌的功能。为探究响应胶孢炭疽菌侵染的柱花草SgMPK6下游互作蛋白,本研究采用酵母双杂交技术,以SgMPK6激酶结构域作为诱饵蛋白,筛选柱花草cDNA文库,共获得74个与SgMPK6激酶结构域潜在的互作蛋白,并通过酵母双杂交点对点试验,验证了候选互作蛋白SgbHLH32、SgbHLH33、SgbHLH44与SgMPK6激酶结构域间的互作关系。磷酸化位点预测显示,3个bHLH转录因子均具有MAPKs潜在的磷酸化位点。柱花草响应炭疽菌侵染的qRT-PCR分析表明,SgbHLH32、SgbHLH33、SgbHLH44转录因子均显著上调,预示互作蛋白可能作为SgMPK6的底物调控柱花草对炭疽病的抗性。本研究为进一步解析SgMPK6响应炭疽菌侵染的分子机制提供了试验依据。

The YDA-MKK4/MKK5-MPK3/MPK6 Cascade Functions Downstream of the RGF1-RGI Ligand-Receptor Pair in Regulating Mitotic Activity in Root Apical Meristem

The mitotic activity of root apical meristem(RAM)is critical to primary root growth and development.Previous studies have identified the roles of ROOT GROWTH FACTOR 1(RGF1),a peptide ligand,and its receptors,RGF1 INSENSITIVEs(RGIs),a clade of five leucine-rich-repeat receptor-like kinases,in promoting cell division in the RAM,which determines the primary root length.However,the downstream signaling components remain elusive.In this study,we identify a complete mitogen-activated protein kinase(MAPK or MPK)cascade,composed of YDA,MKK4/MKK5,and MPK3/MPK6,that functions downstream of the RGF1-RGI ligand-receptor pair.Similar to the rgi1/2/3/4/5 quintuple mutant,loss-of-function mutants of MPK3 and MPK6,MKK4 and MKK5,or YDA show a short-root phenotype,which is associated with reduced mitotic activity and lower expression of PLETHORA 1(PLT1)/PLT2 in the RAM.Furthermore,MPK3/MPK6 activation in response to exogenous RGF1 treatment is impaired in the rgi1/2/3/4/5 quintuple,yda single,and mkk4 m kk5 double mutants.Epistatic analyses demonstrated that the expression of constitutively active MKK4,MKK5,or YDA driven by the RGI2 promoter can rescue the short-root phenotype of the rgi1/2/3/4/5 mutant.Taken together,these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the RGF1-RGI ligand-receptor pair and upstream of PLT1/PLT2 to modulate the stem cell population and primary root growth in Arabidopsis.

A Dominant Allele of Arabidopsis Pectin-Binding Wall-Associated Kinase Induces a Stress Response Suppressed by MPK6 but Not MPK3 Mutations

The plant cell wall is composed of a matrix of cellulose fibers, flexible pectin polymers, and an array of as- sorted carbohydrates and proteins. The receptor-like Wall-Associated Kinases （WAKs） of Arabidopsis bind pectin in the wall, and are necessary both for cell expansion during development and for a response to pathogens and wounding. Mitogen Activated Protein Kinases （MPKs） form a major signaling link between cell surface receptors and both transcrip- tional and enzyme regulation in eukaryotes, and Arabidopsis MPK6 and MPK3 indeed have important roles in develop- ment and the response to stress and pathogens. A dominant allele of WAK2 requires kinase activity and activates a stress response that includes an increased ROS accumulation and the up-regulation of numerous genes involved in pathogen resistance, wounding, and cell wall biogenesis. This dominant allele requires a functional pectin binding and kinase domain, indicating that it is engaged in a WAK signaling pathway. A null mutant of the major plasma membrane ROS-producing enzyme complex, rbohd/f does not suppress the WAK2cTAP-induced phenotype. A mpk6, but not a mpk3, null allele is able to suppress the effects of this dominant WAK2 mutation, thus distinguishing MPK3 and MPK6, whose activity previously was thought to be redundant. Pectin activation of gene expression is abated in a wak2-null, but is tempered by the WAK-dominant allele that induces elevated basal stress-related transcript levels. The results suggest a mechanism in which changes to the cell wall can lead to a large change in cellular responses and help to explain how pathogens and wounding can have general effects on growth.

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development

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)–CRISPRassociated 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.

MYB44 regulates PTI by promoting the expression of EIN2 and MPK3/6 in Arabidopsis

The plant signaling pathway that regulates pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)involves mitogen-activated protein kinase(MAPK)cascades that comprise sequential activation of several protein kinases and the ensuing phosphorylation of MAPKs,which activate transcription factors(TFs)to promote downstream defense responses.To identify plant TFs that regulate MAPKs,we investigated TF-defective mutants of Arabidopsis thaliana and identified MYB44 as an essential constituent of the PTI pathway.MYB44 confers resistance against the bacterial pathogen Pseudomonas syringae by cooperating with MPK3 and MPK6.Under PAMP treatment,MYB44 binds to the promoters of MPK3 and MPK6 to activate their expression,leading to phosphorylation of MPK3 and MPK6 proteins.In turn,phosphorylated MPK3 and MPK6 phosphorylate MYB44 in a functionally redundant manner,thus enabling MYB44 to activate MPK3 and MPK6 expression and further activate downstream defense responses.Activation of defense responses has also been attributed to activation of EIN2 transcription by MYB44,which has previously been shown to affect PAMP recognition and PTI development.AtMYB44 thus functions as an integral component of the PTI pathway by connecting transcriptional and posttranscriptional regulation of the MPK3/6 cascade.

A Kinase-Phosphatase-Transcription Factor Module Regulates Adventitious Root Emergence in Arabidopsis Root-Hypocotyl Junctions

Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely unexplored.Here,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis thaliana.ABI4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR emergence.Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4.MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence.We propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.