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.

Powdery mildew disease resistance and markerassisted screening at the Pm60 locus in wild diploid wheat Triticum urartu

Triticum urartu,a diploid wild wheat and progenitor of the A genome of bread wheat,is an important resource for resistance to powdery mildew fungus caused by Blumeria graminis f.sp.tritici(Bgt).In the present study we systematically characterized the interaction between the Bgt fungus and T.urartu at the microscopic level.We also tested 227 T.urartu accessions for reaction to Bgt isolate E09 and discovered previously uncategorized powdery mildew resistance in this collection.Pm60 is a CC-NB-LRR type powdery mildew resistance gene that has at least three functional alleles,Pm60,Pm60a,and Pm60b.A marker-assisted screen targeting the Pm60 locus identified a non-functional allele of Pm60a,designated as Pm60a′.A sequence comparison of Pm60a′and Pm60a revealed that they differed by 58 SNPs and one 3-nucleotide deletion.Based on the sequence variations two molecular markers were developed to differentiate the functional Pm60a allele from the non-functional Pm60a′.Our screen revealed the presence of a previously uncharacterized powdery mildew resistance in T.urartu and provides new insights into the Pm60 locus.We believe that the two molecular markers developed here and new T.urartu resistant accessions will facilitate further identification of novel powdery mildew resistance genes and benefit breeding for powdery mildew resistance.

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.

Orthologous genesPm12andPm21from twowild relatives of wheat show evolutionary conservation but divergent powdery mildew resistance

Wheat powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is a devastating disease that threatens wheat production worldwide.Pm12,which originated from Aegilops speltoides,a wild relative of wheat,confers strong resistance to powdery mildew and therefore has potential use in wheat breeding.Using susceptible mutants induced by gamma irradiation,we physically mapped and isolated Pm12 and showed it to be orthologous to Pm21 from Dasypyrum villosum,also a wild relative of wheat.The resistance function of Pm12 was validated via ethyl methanesulfonatemutagenesis,virus-induced gene silencing,and stable genetic transformation.Evolutionary analysis indicates that the Pm12/Pm21 loci in wheat species are relatively conserved but dynamic.Here,we demonstrated that the two orthologous genes,Pm12 and Pm21,possess differential resistance against the same set of Bgt isolates.Overexpression of the coiledcoil domains of both PM12 and PM21 induces cell death in Nicotiana benthamiana leaves.However,their full-length forms display different cell death-inducing activities caused by their distinct intramolecular interactions.Cloning of Pm12 will facilitate its application in wheat breeding programs.This study also gives new insight into two orthologous resistance genes,Pm12 and Pm21,which show different race specificities and intramolecular interaction patterns.

Regulation of NLR stability in plant immunity

Plant nucleotide binding domain and leucinerich repeat (NLR) receptors recognize pathogen effectors directly or indirectly and mediate innate immune responses. NLR-mediated immunity also has direct impacts on plant growth and development, as well as yield and survival. The levels of NLR proteins are therefore intricately controlled in plants to balance defense responses and other processes. In recent years, the ubiquitination-26S proteasome system and the HSP90 chaperones have emerged as having key functions in the regulation of NLR stability. The N-end rule pathway of protein degradation is also directly linked to NLR stability. Recent progress in the regulation of NLR stability and turnover is summarized here, focusing on the key components and pathways.

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.