Soybean MKK2 establishes intricate signalling pathways to regulate soybean response to cyst nematode infection.

Mitogen-activated protein kinase (MPK) cascades play central signalling roles in plant immunity and stress response. The soybean orthologue of MPK kinase2 (GmMKK2) was recently identified as a potential signalling node whose expression is upregulated in the feeding site induced by soybean cyst nematode (SCN, Heterodera glycines). To investigate the role of GmMKK2 in soybean-SCN interactions, we overexpressed a catabolically inactive variant referred to as kinase-dead variant (KD-GmMKK2) using transgenic hairy roots. KD-GmMKK2 overexpression caused significant reduction in soybean susceptibility to SCN, while overexpression of the wild-type variant (WT-GmMKK2) exhibited no effect on susceptibility. Transcriptome analysis indicated that KD-GmMKK2 overexpressing plants are primed for SCN resistance via constitutive activation of defence signalling, particularly those related to chitin, respiratory burst, hydrogen peroxide and salicylic acid. Phosphoproteomic profiling of the WT-GmMKK2 and KD-GmMKK2 root samples upon SCN infection resulted in the identification of 391 potential targets of GmMKK2. These targets are involved in a broad range of biological processes, including defence signalling, vesicle fusion, chromatin remodelling and nuclear organization among others. Furthermore, GmMKK2 mediates phosphorylation of numerous transcriptional and translational regulators, pointing to the presence of signalling shortcuts besides the canonical MAPK cascades to initiate downstream signalling that eventually regulates gene expression and translation initiation. Finally, the functional requirement of specific phosphorylation sites for soybean response to SCN infection was validated by overexpressing phospho-mimic and phospho-dead variants of two differentially phosphorylated proteins SUN1 and IDD4. Together, our analyses identify GmMKK2 impacts on signalling modules that regulate soybean response to SCN infection.

The soybean immune receptor GmBIR1 regulates host transcriptome, spliceome, and immunity during cyst nematode infection.

BAK1-INTERACTING RECEPTOR LIKE KINASE1 (BIR1) is a negative regulator of various aspects of disease resistance and immune responses. Here, we investigated the functional role of soybean (Glycine max) BIR1 (GmBIR1) during soybean interaction with soybean cyst nematode (SCN, Heterodera glycines) and the molecular mechanism through which GmBIR1 regulates plant immunity. Overexpression of wild-type variant of GmBIR1 (WT-GmBIR1) using transgenic soybean hairy roots significantly increased soybean susceptibility to SCN, whereas overexpression of kinase-dead variant (KD-GmBIR1) significantly increased plant resistance. Transcriptome analysis revealed that genes oppositely regulated in WT-GmBIR1 and KD-GmBIR1 upon SCN infection were enriched primarily in defense and immunity-related functions. Quantitative phosphoproteomic analysis identified 208 proteins as putative substrates of the GmBIR1 signaling pathway, 114 of which were differentially phosphorylated upon SCN infection. In addition, the phosphoproteomic data pointed to a role of the GmBIR1 signaling pathway in regulating alternative pre-mRNA splicing. Genome-wide analysis of splicing events provided compelling evidence supporting a role of the GmBIR1 signaling pathway in establishing alternative splicing during SCN infection. Our results provide novel mechanistic insights into the function of the GmBIR1 signaling pathway in regulating soybean transcriptome and spliceome via differential phosphorylation of splicing factors and regulation of splicing events of pre-mRNA decay- and spliceosome-related genes.

Establishment and maintenance of DNA methylation in nematode feeding sites.

A growing body of evidence indicates that epigenetic mechanisms, particularly DNA methylation, play key regulatory roles in plant-nematode interactions. Nevertheless, the transcriptional activity of key genes mediating DNA methylation and active demethylation in the nematode feeding sites remains largely unknown. Here, we profiled the promoter activity of 12 genes involved in maintenance and de novo establishment of DNA methylation and active demethylation in the syncytia and galls induced respectively by the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita in Arabidopsis roots. The promoter activity assays revealed that expression of the CG-context methyltransferases is restricted to feeding site formation and development stages. Chromomethylase1 (CMT1), CMT2, and CMT3 and Domains Rearranged Methyltransferase2 (DRM2) and DRM3, which mediate non-CG methylation, showed similar and distinct expression patterns in the syncytia and galls at various time points. Notably, the promoters of various DNA demethylases were more active in galls as compared with the syncytia, particularly during the early stage of infection. Mutants impaired in CG or CHH methylation similarly enhanced plant susceptibility to H. schachtii and M. incognita, whereas mutants impaired in CHG methylation reduced plant susceptibility only to M. incognita. Interestingly, hypermethylated mutants defective in active DNA demethylation exhibited contrasting responses to infection by H. schachtii and M. incognita, a finding most likely associated with differential regulation of defense-related genes in these mutants upon nematode infection. Our results point to methylation-dependent mechanisms regulating plant responses to infection by cyst and root-knot nematodes.