HvWRKY2 acts as an immunity suppressor and targets HvCEBiP to regulate powdery mildew resistance in barley

Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.

HvMPK4 phosphorylates HvWRKY1 to enhance its suppression of barley immunity to powdery mildew fungus

Mitogen-activated protein kinase(MAPK)cascades play important roles in disease resistance in model plant species.However,the functions of MAPK signaling pathways in crop disease resistance are largely unknown.Here we report the function of HvMKK1-HvMPK4-HvWRKY1 module in barley immune system.HvMPK4 is identified to play a negative role in barley immune response against Bgh,as virus-induced gene silencing of HvMPK4 results in enhanced disease resistance whilst stably overexpressing HvMPK4 leads to super-susceptibility to Bgh infection.Furthermore,the barley MAPK kinase HvMKK1 is found to specifically interact with HvMPK4,and the activated HvMKK1^(DD) variant specifically phosphorylates HvMPK4 in vitro.Moreover,the transcription factor HvWRKY1 is identified to be a downstream target of HvMPK4 and phosphorylated by HvMPK4 in vitro in the presence of HvMKK1^(DD).Phosphorylation assay coupled with mutagenesis analyses identifies S122,T284,and S347 in HvWRKY1 as the major residues phosphorylated by HvMPK4.HvWRKY1 is phosphorylated in barley at the early stages of Bgh infection,which enhances its suppression on barley immunity likely due to enhanced DNA-binding and transcriptional repression activity.Our data suggest that the HvMKK1-HvMPK4 kinase pair acts upstream of HvWRKY1 to negatively regulate barley immunity against powdery mildew.

SnRK1 Phosphorylates and Destabilizes WRKY3 to Enhance Barley Immunity to Powdery Mildew

Plants recognize pathogens and activate immune responses,which usually involve massive transcriptional reprogramming.The evolutionarily conserved kinase,Sucrose non-fermenting-related kinase 1(SnRK1),functions as a metabolic regulator that is essential for plant growth and stress responses.Here,we identify barley SnRK1 and a WRKY3 transcription factor by screening a cDNA library.SnRK1 interacts with WRKY3 in yeast,as confirmed by pull-down and luciferase complementation assays.Förster resonance energy transfer combined with noninvasive fluorescence lifetime imaging analysis indicates that the interaction occurs in the barley nucleus.Transient expression and virus-induced gene silencing analyses indicate that WRKY3 acts as a repressor of disease resistance to the Bgh fungus.Barley plants overexpressing WRKY3 have enhanced fungal microcolony formation and sporulation.Phosphorylation assays show that SnRK1 phosphorylates WRKY3 mainly at Ser83 and Ser112 to destabilize the repressor,and WRKY3 non-phosphorylation-null mutants at these two sites are more stable than the wild-type protein.SnRK1-overexpressing barley plants display enhanced disease resistance to Bgh.Transient expression of SnRK1 reduces fungal haustorium formation in barley cells,which probably requires SnRK1 nuclear localization and kinase activity.Together,these findings suggest that SnRK1 is directly involved in plant immunity through phosphorylation and destabilization of the WRKY3 repressor,revealing a new regulatory mechanism of immune derepression in plants.