Overexpression of alfalfa SIMK promotes root hair growth, nodule clustering and shoot biomass production

Hrbáčková, Miroslava; Luptovčiak, Ivan; Hlaváčková, Kateřina; Dvořák, Petr; Tichá, Michaela; Šamajová, Olga; Novák, Dominik; Bednarz, Hanna; Niehaus, Karsten; Ovečka, Miroslav; Šamaj, Jozef

doi:10.1111/pbi.13503

Cited by 10 publications

(23 citation statements)

References 74 publications

(94 reference statements)

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“…Analysis of hairy root transformed plants demonstrate a non-phosphorylatable mutant LjMPK6 (T224A Y226F) mimics LjPP2C, illustrating LjPP2C phosphatase dephosphorylates LjMPK6 to fine tune nodule development in L. japonicus (Yan et al, 2020 ). The upstream SIMK activator SIMK kinase (SIMKK) infected with Sinorhizobium meliloti show in SIMKK RNAi lines exhibiting downregulation of both SIMKK and SIMK, having reduced root hair growth while also having a decrease in ability to form infection threads and nodules (Hrbáčková et al, 2021 ). Constitutive SIMK OE promotes root hair growth, infection thread, and nodule clustering (Hrbáčková et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…The upstream SIMK activator SIMK kinase (SIMKK) infected with Sinorhizobium meliloti show in SIMKK RNAi lines exhibiting downregulation of both SIMKK and SIMK, having reduced root hair growth while also having a decrease in ability to form infection threads and nodules (Hrbáčková et al, 2021 ). Constitutive SIMK OE promotes root hair growth, infection thread, and nodule clustering (Hrbáčková et al, 2021 ). The results are consistent with the involvement of DMI genes in G. max in AM symbioses (Navazio et al, 2007 ).…”

Section: Discussionmentioning

confidence: 99%

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Glycine max Homologs of DOESN'T MAKE INFECTIONS 1, 2, and 3 Function to Impair Heterodera glycines Parasitism While Also Regulating Mitogen Activated Protein Kinase Expression

et al. 2022

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Glycine max root cells developing into syncytia through the parasitic activities of the pathogenic nematode Heterodera glycines underwent isolation by laser microdissection (LM). Microarray analyses have identified the expression of a G. max DOESN'T MAKE INFECTIONS3 (DMI3) homolog in syncytia undergoing parasitism but during a defense response. DMI3 encodes part of the common symbiosis pathway (CSP) involving DMI1, DMI2, and other CSP genes. The identified DMI gene expression, and symbiosis role, suggests the possible existence of commonalities between symbiosis and defense. G. max has 3 DMI1, 12 DMI2, and 2 DMI3 paralogs. LM-assisted gene expression experiments of isolated syncytia under further examination here show G. max DMI1-3, DMI2-7, and DMI3-2 expression occurring during the defense response in the H. glycines-resistant genotypes G.max[Peking/PI548402] and G.max[PI88788] indicating a broad and consistent level of expression of the genes. Transgenic overexpression (OE) of G. max DMI1-3, DMI2-7, and DMI3-2 impairs H. glycines parasitism. RNA interference (RNAi) of G. max DMI1-3, DMI2-7, and DMI3-2 increases H. glycines parasitism. The combined opposite outcomes reveal a defense function for these genes. Prior functional transgenic analyses of the 32-member G. max mitogen activated protein kinase (MAPK) gene family has determined that 9 of them act in the defense response to H. glycines parasitism, referred to as defense MAPKs. RNA-seq analyses of root RNA isolated from the 9 G. max defense MAPKs undergoing OE or RNAi reveal they alter the relative transcript abundances (RTAs) of specific DMI1, DMI2, and DMI3 paralogs. In contrast, transgenically-manipulated DMI1-3, DMI2-7, and DMI3-2 expression influences MAPK3-1 and MAPK3-2 RTAs under certain circumstances. The results show G. max homologs of the CSP, and defense pathway are linked, apparently involving co-regulated gene expression.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Glycine max Homologs of DOESN'T MAKE INFECTIONS 1, 2, and 3 Function to Impair Heterodera glycines Parasitism While Also Regulating Mitogen Activated Protein Kinase Expression

et al. 2022

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“…Considering MAPK developmental roles in root hair formation, these are known solely for Arabidopsis MPK4 ( Beck et al, 2010 ) and Medicago sativa SIMK, which is an orthologue of Arabidopsis MPK6 ( Šamaj et al, 2002 ; Hrbáčková et al, 2020 , in press) so far. This study is the first one reporting about the involvement of MPK3 in root hair growth.…”

Section: Discussionmentioning

confidence: 99%

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley

et al. 2021

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Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

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“…Alfalfa wild-type plants of cv. Regen-SY (RSY) carrying either 35S::GFP:SIMK construct (GFP-SIMK fusion protein; Hrbáčková et al, 2021) or SIMKK-RNAi in pHellsgate12 plasmid driven under 35S promoter (obtained from CSIRO Plant Industry, Australia) were obtained by regeneration in vitro through somatic embryogenesis from leaf explants as previously described (Samac and Austin-Phillips, 2006;Bekešová et al, 2015;Hrbáčková et al, 2021).…”

Section: Plant Materialsmentioning

confidence: 99%

“…Regenerated alfalfa plants RSY, transgenic SIMKK-RNAi line (showing strong downregulation of SIMKK and SIMK transcripts and SIMK protein), and transgenic GFP-SIMK line with upregulated SIMK transcript and enhanced SIMK activity (Hrbáčková et al, 2021) were transferred to nitrogen-free Fåhreus medium (FAH-N 2 ; Fåhreus, 1957) for inoculation with rhizobia.…”

Section: Plant Materialsmentioning

confidence: 99%

Genetic manipulation of stress-induced mitogen-activated protein kinase modulates early stages of the nodulation process inMedicago sativa

Ovečka

Hlaváčková

Šamajová

et al. 2022

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Leguminous plants have established a mutualistic endosymbiotic interaction with nitrogen-fixing rhizobia to secure nitrogen sources in new specialized organs called root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We have recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule and shoot formation. However, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during the early stages of alfalfa nodulation remain unclear. Here, we qualitatively and quantitatively characterized SIMK distribution patterns in rhizobium-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In theSIMKK-RNAiline, showing downregulation ofSIMKKandSIMK, we found a considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads, which was strongly increased in the GFP-SIMK line, constitutively overexpressing GFP-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. These results shed new light on SIMK spatio-temporal participation in the early interactions between alfalfa and rhizobia, and its internalization into root hairs, showing that local accumulation of active SIMK indeed modulates nodulation in alfalfa.One sentence summaryGenetic manipulation of SIMK in alfalfa revealed that SIMK modulates root hair capacity to form infection pockets and infection threads during the early interactions between alfalfa and rhizobia.

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Overexpression of alfalfa SIMK promotes root hair growth, nodule clustering and shoot biomass production

Cited by 10 publications

References 74 publications

Glycine max Homologs of DOESN'T MAKE INFECTIONS 1, 2, and 3 Function to Impair Heterodera glycines Parasitism While Also Regulating Mitogen Activated Protein Kinase Expression

Glycine max Homologs of DOESN'T MAKE INFECTIONS 1, 2, and 3 Function to Impair Heterodera glycines Parasitism While Also Regulating Mitogen Activated Protein Kinase Expression

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley

Genetic manipulation of stress-induced mitogen-activated protein kinase modulates early stages of the nodulation process inMedicago sativa

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