Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

Structural polymorphisms within a common powdery mildew effector scaffold as a driver of coevolution with cereal immune receptors.

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.

A fungal plant pathogen overcomes mlo-mediated broad-spectrum disease resistance by rapid gene loss.

Hosts and pathogens typically engage in a coevolutionary arms race. This also applies to phytopathogenic powdery mildew fungi, which can rapidly overcome plant resistance and perform host jumps. Using experimental evolution, we show that the powdery mildew pathogen Blumeria hordei is capable of breaking the agriculturally important broad-spectrum resistance conditioned by barley loss-of-function mlo mutants. Partial mlo virulence of evolved B. hordei isolates is correlated with a distinctive pattern of adaptive mutations, including small-sized (c. 8-40 kb) deletions, of which one is linked to the de novo insertion of a transposable element. Occurrence of the mutations is associated with a transcriptional induction of effector protein-encoding genes that is absent in mlo-avirulent isolates on mlo mutant plants. The detected mutational spectrum comprises the same loci in at least two independently isolated mlo-virulent isolates, indicating convergent multigenic evolution. The mutational events emerged in part early (within the first five asexual generations) during experimental evolution, likely generating a founder population in which incipient mlo virulence was later stabilized by additional events. This work highlights the rapid dynamic genome evolution of an obligate biotrophic plant pathogen with a transposon-enriched genome.

Long-term and rapid evolution in powdery mildew fungi.

The powdery mildew fungi (Erysiphaceae) are globally distributed plant pathogens with a range of more than 10,000 plant hosts. In this review, we discuss the long- and short-term evolution of these obligate biotrophic fungi and outline their diversity with respect to morphology, lifestyle, and host range. We highlight their remarkable ability to rapidly overcome plant immunity, evolve fungicide resistance, and broaden their host range, for example, through adaptation and hybridization. Recent advances in genomics and proteomics, particularly in cereal powdery mildews (genus Blumeria), provided first insights into mechanisms of genomic adaptation in these fungi. Transposable elements play key roles in shaping their genomes, where even close relatives exhibit diversified patterns of recent and ongoing transposon activity. These transposons are ubiquitously distributed in the powdery mildew genomes, resulting in a highly adaptive genome architecture lacking obvious regions of conserved gene space. Transposons can also be neofunctionalized to encode novel virulence factors, particularly candidate secreted effector proteins, which may undermine the plant immune system. In cereals like barley and wheat, some of these effectors are recognized by plant immune receptors encoded by resistance genes with numerous allelic variants. These effectors determine incompatibility ("avirulence") and evolve rapidly through sequence diversification and copy number variation. Altogether, powdery mildew fungi possess plastic genomes that enable their fast evolutionary adaptation towards overcoming plant immunity, host barriers, and chemical stress such as fungicides, foreshadowing future outbreaks, host range shifts and expansions as well as potential pandemics by these pathogens.

Long noncoding RNAs emerge from transposon-derived antisense sequences and may contribute to infection stage-specific transposon regulation in a fungal phytopathogen.

BACKGROUND: The genome of the obligate biotrophic phytopathogenic barley powdery mildew fungus Blumeria hordei is inflated due to highly abundant and possibly active transposable elements (TEs). In the absence of the otherwise common repeat-induced point mutation transposon defense mechanism, noncoding RNAs could be key for regulating the activity of TEs and coding genes during the pathogenic life cycle. RESULTS: We performed time-course whole-transcriptome shotgun sequencing (RNA-seq) of total RNA derived from infected barley leaf epidermis at various stages of fungal pathogenesis and observed significant transcript accumulation and time point-dependent regulation of TEs in B. hordei. Using a manually curated consensus database of 344 TEs, we discovered phased small RNAs mapping to 104 consensus transposons, suggesting that RNA interference contributes significantly to their regulation. Further, we identified 5,127 long noncoding RNAs (lncRNAs) genome-wide in B. hordei, of which 823 originated from the antisense strand of a TE. Co-expression network analysis of lncRNAs, TEs, and coding genes throughout the asexual life cycle of B. hordei points at extensive positive and negative co-regulation of lncRNAs, subsets of TEs and coding genes. CONCLUSIONS: Our work suggests that similar to mammals and plants, fungal lncRNAs support the dynamic modulation of transcript levels, including TEs, during pivotal stages of host infection. The lncRNAs may support transcriptional diversity and plasticity amid loss of coding genes in powdery mildew fungi and may give rise to novel regulatory elements and virulence peptides, thus representing key drivers of rapid evolutionary adaptation to promote pathogenicity and overcome host defense.

Clumpy artifacts can be differentiated from tophi with DECT: comparison between gout-free and gouty patients.

OBJECTIVES: To accurately differentiate clumpy artifacts from tophi with foot and ankle DECT. METHODS AND MATERIALS: In session 1, 108 clumpy artifacts from 35 patients and 130 tophi images from 25 patients were analyzed. Reviewers classified green pixelation according to anatomic location, shape (linear, stippled, angular, oval), and height and width ratio. In session 2, green pixelation confined to the tendon was evaluated (shape, height and width ratio, occupied area in the tendon, accompanied peritendinous green pixelation). RESULTS: In session 1, while tophi were noted at various locations, almost all clumpy artifacts were located at the tendon (99%, p < 0.0001). Most clumpy artifacts were linear, stippled, and wide, while most tophi were angular and oval (p < 0.05). In session 2, the shape of green pixelation from clumpy artifacts and tophi was significantly different (p < 0.0001) and most clumpy artifacts occupied less than 50% of the tendon (p = 0.02), and most tophi were accompanied by peritendinous green pixelation (p < 0.0001). Univariant logistic regression showed that tophi were significantly correlated with peritendinous deposits, angular and oval shape, and more than 50% of the tendon (p < 0.05). CONCLUSION: Clumpy artifacts can be differentiated from tophi in DECT. Clumpy artifacts typically are located in the tendon with a linear or stippled shape, wide, and less than 50% of a tendon's cross-section. Tophi, on the other hand, typically are oval, larger than 50% of the tendon's cross-section, and associated with adjacent peritendinous green pixelation. ADVANCES IN KNOWLEDGE: Clumpy artifacts can be differentiated from tophi in image findings by their location and shape.

Multiple pairs of allelic MLA immune receptor-powdery mildew AVRA effectors argue for a direct recognition mechanism.

Nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins in plants and animals mediate intracellular pathogen sensing. Plant NLRs typically detect strain-specific pathogen effectors and trigger immune responses often linked to localized host cell death. The barley Mla disease resistance locus has undergone extensive functional diversification in the host population and encodes numerous allelic NLRs each detecting a matching isolate-specific avirulence effector (AVRA) of the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). We report here the isolation of Bgh AVRa7, AVRa9, AVRa10, and AVRa22, which encode small secreted proteins recognized by allelic MLA7, MLA9, MLA10, and MLA22 receptors, respectively. These effectors are sequence-unrelated, except for allelic AVRa10 and AVRa22 that are co-maintained in pathogen populations in the form of a balanced polymorphism. Contrary to numerous examples of indirect recognition of bacterial effectors by plant NLRs, co-expression experiments with matching Mla-AVRa pairs indicate direct detection of the sequence-unrelated fungal effectors by MLA receptors.

Metacognitive Confidence Increases with, but Does Not Determine, Visual Perceptual Learning.

While perceptual learning increases objective sensitivity, the effects on the constant interaction of the process of perception and its metacognitive evaluation have been rarely investigated. Visual perception has been described as a process of probabilistic inference featuring metacognitive evaluations of choice certainty. For visual motion perception in healthy, naive human subjects here we show that perceptual sensitivity and confidence in it increased with training. The metacognitive sensitivity-estimated from certainty ratings by a bias-free signal detection theoretic approach-in contrast, did not. Concomitant 3Hz transcranial alternating current stimulation (tACS) was applied in compliance with previous findings on effective high-low cross-frequency coupling subserving signal detection. While perceptual accuracy and confidence in it improved with training, there were no statistically significant tACS effects. Neither metacognitive sensitivity in distinguishing between their own correct and incorrect stimulus classifications, nor decision confidence itself determined the subjects' visual perceptual learning. Improvements of objective performance and the metacognitive confidence in it were rather determined by the perceptual sensitivity at the outset of the experiment. Post-decision certainty in visual perceptual learning was neither independent of objective performance, nor requisite for changes in sensitivity, but rather covaried with objective performance. The exact functional role of metacognitive confidence in human visual perception has yet to be determined.