rcs5-mediated spot blotch resistance in barley is conferred by wall-associated kinases that resist pathogen manipulation

Abstract

27Plant biotrophic pathogen disease resistances rely on immunity receptor-mediated programmed 28 cell death (PCD) responses, but specialized necrotrophic/hemi-biotrophic pathogens hijack these 29 mechanisms to colonize the resulting dead tissue in their necrotrophic phase. Thus, immunity receptors 30 can become necrotrophic pathogen dominant susceptibility targets but resistance mechanisms that resist 31 necrotroph manipulation are recessive resistance genes. The barley rcs5 QTL imparts recessive resistance 32 against the disease spot blotch caused by the hemi-biotrophic fungal pathogen Bipolaris sorokiniana. The 33 rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four 34 wall-associated kinase (WAK) genes, designated HvWak2, Sbs1, Sbs2 (susceptibility to Bipolaris 35 sorokiniana 1&2), and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in susceptible barley 36 cultivars resulted in resistance showing dominant susceptibility function. Allele analysis of Sbs1&2 from 37 resistant and susceptible barley cultivars identified sequence polymorphisms associated with phenotypes 38in their primary coding sequence and promoter regions, suggesting differential transcriptional regulation 39 may contribute to susceptibility. Transcript analysis of Sbs1&2 showed nearly undetectable expression in 40 resistant and susceptible cultivars prior to pathogen challenge; however, upregulation of both genes 41 occurred specifically in susceptible cultivars post-inoculation with a virulent isolate. Apoplastic wash 42 fluids collected from barley infected with a virulent isolate induced Sbs1, suggesting regulation by an 43 apoplastic-secreted effector. Thus, Sbs1&2 function as B. sorokiniana susceptibility targets and non-44 functional alleles or alleles that resist induction by the pathogen mediate rcs5-recessive resistance. The 45 sbs1&2 alleles underlying the rcs5 QTL that the pathogen is unable to manipulate are the first resistance 46 genes identified against spot blotch. 47Bipolaris sorokiniana 1 and 2) as wall-associated kinases. These genes are hijacked by the 53 hemibiotrophic pathogen in its necrotrophic phase to induce programmed cell death, facilitating disease 54 development. We report the first spot blotch resistance/susceptibility genes cloned that function via alleles 55 that cannot be specifically induced and hijacked by virulent isolates of the pathogen. 56 study on a large breeding panel provided a comprehensive assessment of the genetic architecture of this 79 durable spot blotch resistance, identifying three quantitative trait loci (QTL): Rcs-qtl-1H-11_10764, Rcs-80 qtl-3H-11_10565 and Rcs-qtl-7H-11_20162. Of these QTL, Rcs-qtl-7H-11_20162 conferred the largest 81 allelic effect across different populations (10) with several genetic studies positioning rcs5 within the 82 interval of this chromosome 7H QTL (9-11). Thus, the gene/s underlying the rcs5 QTL is an important 83 target for cloning and functional characterization, which was the objective of this study. 8...

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability

“…This aberrant interaction possibly lead to the rapid ROS production in the nec3 mutants and the runaway cell death due to the lack of serotonin accumulation which may play a role in the sequestration of PCD mediated lesion expansion similar to the sl mutant in rice. The DAB assays supported this conclusion as the nec3-γ1 mutant produced ROS when infected by the necrotrophic pathogen B. sorokiniana as early as 12 hpi as compared to a delayed ROS in the susceptible Steptoe and resistant Bowman which was detected at 18 and 24 hpi, respectively (19) (Figure 4).…”

“…The DAMPs are host derived extracellular matrix subunits such as oligogalacturonide residues (OGs) released from plant cell walls upon partial degradation by pathogens. The detection of OGs by wall associated kinase (WAK) PRRs sound the alarm of compromised cell integrity, indicating pathogen ingress or challenge eliciting the DTI defense responses (18,19).…”

Towards Stewardship of Wild Species and Their Domesticated Counterparts: A Case Study in Northern Wild Rice (Zizania palustrisL.)

“…Eleven genes specifically related to drought stress and abscisic acid (ABA) signaling in other species included the following: a calmodulin-binding receptor-like cytoplasmic kinase 3 (Yang et al, 2010; Sun et al, 2021) on ZPchr0004; a calcium-dependent protein kinase family protein (Campo et al, 2014; Wei et al, 2014) on ZPchr0005; two copies of coatomer subunit beta’-1 isoform X1 (Mirzaei et al, 2014), a zinc finger CCCH domain-containing protein 43 (Peng et al, 2012), and a receptor-like protein kinase At5g2005 (Marshall et al, 2012) on ZPchr0006; a CBL-interacting protein kinase 32 (Tai et al, 2016) on ZPchr0011; two copies of ras-related protein RABC2a (Mérida-Garcia et al, 2020) and E3 ubiquitin-protein ligase RZFP34 isoform X2 (Ding et al, 2015; Shu and Yang, 2017) on ZPchr0013; methyltransferase-like protein 13 isoform X2 (Gupta et al, 2020) on ZPchr0015. We also identified five genes in these regions known to confer disease resistance in other species including a LRR receptor-like serine/threonine-protein kinase At1g06840 isoform X1 (Kumar et al, 2018; Guo et al, 2022), a respiratory burst oxidase homolog protein H (Sagi et al, 2004; Wang et al, 2018), a hydrolase, NUDIX family protein (Bartsch et al, 2006; Ge and Xia, 2008), a 60S ribosomal protein L3 (Di et al, 2010; Kant et al, 2012), and the barley B recombinant-like protein A (Ameen et al, 2020).…”

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability

“…sorokiniana isolate ND85F. After detachment, the leaves were immediately transferred to 10 ml of freshly prepared 1mg/ml DAB (Sigma Aldrich, MO) solution (pH 3.6) in 15 ml tubes following the protocol described by [ 53 ].…”

“…To observe ROS at the site of infection, five 3 cm leaf samples were collected from secondary leaves at 6, 12, 18, 24 and 48 hpi from WT Bowman and nec3-γ1 mutant seedlings inoculated with B. sorokiniana isolate ND85F. After detachment, the leaves were immediately transferred to 10 ml of freshly prepared 1mg/ml DAB (Sigma Aldrich, MO) solution (pH 3.6) in 15 ml tubes following the protocol described by [53].…”