An active form of Jasmonate Induced Protein 60 (JIP60) from Hordeum vulgare cv. Golden Promise

Jasmonate Induced protein 60 (JIP60) was amplified from barley leaves infected with Blumeria graminis (Pennington et al., 2018). A peptide N-terminal domain was excised, and replaced with either a methionine-leucine (ML) linker, or a methionine, leucine, aspartic acid & proline linker (MLDP) to produce active forms of the JIP60 protein, Figure 1. Mak et al., (2007) had previously shown that these modifications were required to produce an active maize ribosome-inactivating protein (b-32). The Gateway entry construct pDONR_JIP60mldp is available from Addgene https://www.addgene.org/104959

The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus

Structural basis for the reduced affinity of mfH with fHbp.

<p>(<b>A</b>) Cartoon of hfH₆₇ viewed from through V1 fHbp (solid line) with amino acids changed in hfH with murine residues (outlined by yellow dashes), and those replaced in mfH with human residues (outlined by light blue dashes). (<b>B</b>) SPR analysis of binding of two hfH₆₇ mutants each containing two amino acid changes (shown) with fHbps from each variant family. (<b>C</b>) Far western analysis of V1 fHbp and a control protein, PPX; blots were overlaid with 5 µg/ml of the recombinant proteins mfH, modified mfH (with 14 humanised amino acids) or hfH, or with human serum (1 in 2000 dilution) as indicated; the sizes of the mol. wt. marker are shown. (<b>D</b>) Structure of mfH₆₇ (blue ribbon) superimposed on V1 fHbp (white ribbon) and hfH (green ribbon). While fH₆ from both species are superimposable, the orientation of fH₇ differs significantly between mfH and hfH (indicated in red dashed circle).</p

Non-functional fHbps retain their immunogenicity in transgenic mice.

<p>(<b>A</b>) ELISAs assaying anti-V1 titres elicited in pooled sera following immunisation of transgenic mice with the wild-type protein and non-functional V1 fHbps. (<b>B</b>) SBA titres of sera from individual mice immunisation with fHbps.</p

Effect of mutations at positions equivalent to fHbp V1 residues 283 and 304 (<i>i.e.</i> Thr³⁰⁴ in V2 and V3 fHbp) on the <i>K</i>_D for binding to fH₆₇, shown relative to the wild-type proteins.

<p>DM indicates double Ala substitution; ND, not determined.</p

Brecciation at the grain scale within the lithologies of the Winchcombe Mighei‐like carbonaceous chondrite

The Mighei‐like carbonaceous (CM) chondrites have been altered to various extents by water–rock reactions on their parent asteroid(s). This aqueous processing has destroyed much of the primary mineralogy of these meteorites, and the degree of alteration is highly heterogeneous at both the macroscale and nanoscale. Many CM meteorites are also heavily brecciated juxtaposing clasts with different alteration histories. Here we present results from the fine‐grained team consortium study of the Winchcombe meteorite, a recent CM chondrite fall that is a breccia and contains eight discrete lithologies that span a range of petrologic subtypes (CM2.0–2.6) that are suspended in a cataclastic matrix. Coordinated multitechnique, multiscale analyses of this breccia reveal substantial heterogeneity in the extent of alteration, even in highly aqueously processed lithologies. Some lithologies exhibit the full range and can comprise nearly unaltered coarse‐grained primary components that are found directly alongside other coarse‐grained components that have experienced complete pseudomorphic replacement by secondary minerals. The preservation of the complete alteration sequence and pseudomorph textures showing tochilinite–cronstedtite intergrowths are replacing carbonates suggest that CMs may be initially more carbonate rich than previously thought. This heterogeneity in aqueous alteration extent is likely due to a combination of microscale variability in permeability and water/rock ratio generating local microenvironments as has been established previously. Nevertheless, some of the disequilibrium mineral assemblages observed, such as hydrous minerals juxtaposed with surviving phases that are typically more fluid susceptible, can only be reconciled by multiple generations of alteration, disruption, and reaccretion of the CM parent body at the grain scale.</p