Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation.

The nuclear pore complex (NPC) is vital for nucleocytoplasmic communication. Recent evidence emphasizes its extensive association with proteins of diverse functions, suggesting roles beyond cargo transport. Yet, our understanding of NPC's composition and functionality at this extended level remains limited. Here, through proximity-labelling proteomics, we uncover both local and global NPC-associated proteome in Arabidopsis, comprising over 500 unique proteins, predominantly associated with NPC's peripheral extension structures. Compositional analysis of these proteins revealed that the NPC concentrates chromatin remodellers, transcriptional regulators and mRNA processing machineries in the nucleoplasmic region while recruiting translation regulatory machinery on the cytoplasmic side, achieving a remarkable orchestration of the genetic information flow by coupling RNA transcription, maturation, transport and translation regulation. Further biochemical and structural modelling analyses reveal that extensive interactions with nucleoporins, along with phase separation mediated by substantial intrinsically disordered proteins, may drive the formation of the unexpectedly large nuclear pore proteome assembly.

Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Altering Specificity and Autoactivity of Plant Immune Receptors Sr33 and Sr50 Via a Rational Engineering Approach.

Many resistance genes deployed against pathogens in crops are intracellular nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs). The ability to rationally engineer the specificity of NLRs will be crucial in the response to newly emerging crop diseases. Successful attempts to modify NLR recognition have been limited to untargeted approaches or depended on previously available structural information or knowledge of pathogen-effector targets. However, this information is not available for most NLR-effector pairs. Here, we demonstrate the precise prediction and subsequent transfer of residues involved in effector recognition between two closely related NLRs without their experimentally determined structure or detailed knowledge about their pathogen effector targets. By combining phylogenetics, allele diversity analysis, and structural modeling, we successfully predicted residues mediating interaction of Sr50 with its cognate effector AvrSr50 and transferred recognition specificity of Sr50 to the closely related NLR Sr33. We created synthetic versions of Sr33 that contain amino acids from Sr50, including Sr33syn, which gained the ability to recognize AvrSr50 with 12 amino-acid substitutions. Furthermore, we discovered that sites in the LRR domain needed to transfer recognition specificity to Sr33 also influence autoactivity in Sr50. Structural modeling suggests these residues interact with a part of the NB-ARC domain, which we named the NB-ARC latch, to possibly maintain the inactive state of the receptor. Our approach demonstrates rational modifications of NLRs, which could be useful to enhance existing elite crop germplasm. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Paralog editing tunes rice stomatal density to maintain photosynthesis and improve drought tolerance.

Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. We explored stomatal developmental genetics as a mechanism for improving drought resilience in rice while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of the positive regulator of stomatal development STOMAGEN and its paralog EPIDERMAL PATTERNING FACTOR-LIKE10 (EPFL10) yielded lines with ∼25% and 80% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal density were able to conserve water to similar extents as stomagen lines but did not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal density achieved by editing EPFL10 present a climate-adaptive approach for safeguarding yield in rice. Editing the paralog of STOMAGEN in other species may provide a means for tuning stomatal density in agriculturally important crops beyond rice.

Prediction of effector protein structures from fungal phytopathogens enables evolutionary analyses.

Elucidating the similarity and diversity of pathogen effectors is critical to understand their evolution across fungal phytopathogens. However, rapid divergence that diminishes sequence similarities between putatively homologous effectors has largely concealed the roots of effector evolution. Here we modelled the structures of 26,653 secreted proteins from 14 agriculturally important fungal phytopathogens, six non-pathogenic fungi and one oomycete with AlphaFold 2. With 18,000 successfully predicted folds, we performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded sequence-unrelated structurally similar (SUSS) effector families and common folds present across the fungal species. Extreme expansion of lineage-specific SUSS effector families was found only in several obligate biotrophs, Blumeria graminis and Puccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified new classes of SUSS effector families that include known virulence factors, such as AvrSr35, AvrSr50 and Tin2. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Putatively sub- and neo-functionalized SUSS effectors could reconverge on regulation, expanding the functional pools of effectors in the pathogen infection cycle. We also found evidence that many effector families could have originated from ancestral folds conserved across fungi. Collectively, our study highlights diverse effector evolution mechanisms and supports divergent evolution as a major force in driving SUSS effector evolution from ancestral proteins.

Diversity of genomic adaptations to the post-fire environment in Pezizales fungi points to crosstalk between charcoal tolerance and sexual development.

Wildfires drastically impact the soil environment, altering the soil organic matter, forming pyrolyzed compounds, and markedly reducing the diversity of microorganisms. Pyrophilous fungi, especially the species from the orders Pezizales and Agaricales, are fire-responsive fungal colonizers of post-fire soil that have historically been found fruiting on burned soil and thus may encode mechanisms of processing these compounds in their genomes. Pyrophilous fungi are diverse. In this work, we explored this diversity and sequenced six new genomes of pyrophilous Pezizales fungi isolated after the 2013 Rim Fire near Yosemite Park in California, USA: Pyronema domesticum, Pyronema omphalodes, Tricharina praecox, Geopyxis carbonaria, Morchella snyderi, and Peziza echinospora. A comparative genomics analysis revealed the enrichment of gene families involved in responses to stress and the degradation of pyrolyzed organic matter. In addition, we found that both protein sequence lengths and G + C content in the third base of codons (GC3) in pyrophilous fungi fall between those in mesophilic/nonpyrophilous and thermophilic fungi. A comparative transcriptome analysis of P. domesticum under two conditions - growing on charcoal, and during sexual development - identified modules of genes that are co-expressed in the charcoal and light-induced sexual development conditions. In addition, environmental sensors such as transcription factors STE12, LreA, LreB, VosA, and EsdC were upregulated in the charcoal condition. Taken together, these results highlight genomic adaptations of pyrophilous fungi and indicate a potential connection between charcoal tolerance and fruiting body formation in P. domesticum.

Computational Structural Genomics Unravels Common Folds and Novel Families in the Secretome of Fungal Phytopathogen Magnaporthe oryzae.

Structural biology has the potential to illuminate the evolution of pathogen effectors and their commonalities that cannot be readily detected at the primary sequence level. Recent breakthroughs in protein structure modeling have demonstrated the feasibility to predict the protein folds without depending on homologous templates. These advances enabled a genome-wide computational structural biology approach to help understand proteins based on their predicted folds. In this study, we employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, we predicted the folds of 1,295 proteins (70%). We showed that template-free modeling by TrRosetta captured 514 folds missed by homology modeling, including many known effectors and virulence factors, and that TrRosetta generally produced higher quality models for secreted proteins. Along with sensitive homology search, we employed structure-based clustering, defining not only homologous groups with divergent members but also sequence-unrelated structurally analogous groups. We demonstrate that this approach can reveal new putative members of structurally similar MAX effectors and novel analogous effector families present in M. oryzae and possibly in other phytopathogens. We also investigated the evolution of expanded putative ADP-ribose transferases with predicted structures. We suggest that the loss of catalytic activities of the enzymes might have led them to new evolutionary trajectories to be specialized as protein binders. Collectively, we propose that computational structural genomics approaches can be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of machine learning-based structure prediction.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Functional specificity, diversity, and redundancy of Arabidopsis JAZ family repressors in jasmonate and COI1-regulated growth, development, and defense.

In response to jasmonates (JAs), the JA receptor Coronatine Insensitive 1 (COI1) recruits JA-zinc-finger inflorescence meristem (ZIM)-domain (JAZ) family repressors for destruction to regulate plant growth, development, and defense. As Arabidopsis encodes 13 JAZ repressors, their functional specificity, diversity, and redundancy in JA/COI1-mediated responses remain unclear. We generated a broad range of jaz mutants based on their phylogenetic relationship to investigate their roles in JA responses. The group I JAZ6 may play an inhibitory role in resistance to Botrytis cinerea, group II (JAZ10)/III (JAZ11/12) in JA-regulated root growth inhibition and susceptibility to Pseudomonas syringae pv tomato DC3000, and group IV JAZ3/4/9 in flowering time delay and defense against insects. JAZs exhibit high redundancy in apical hook curvature. The undecuple jaz1/2/3/4/5/6/7/9/10/11/12 (jaz1-7,9-12) mutations enhance JA responses and suppress the phenotypes of coi1-1 in flowering time, rosette growth, and defense. The JA hypersensitivity of jaz1-7,9-12 in root growth, hook curvature, and leaf yellowing is blocked by coi1-1. jaz1-7,9-12 does not influence the stamen phenotypes of wild-type and coi1-1. jaz1-7,9-12 affects JA-regulated transcriptional profile and recovers a fraction of that in coi1-1. This study contributes to elucidating the specificity, diversity, and redundancy of JAZ members in JA/COI1-regulated growth, development, and defense responses.

Evolution of NLR resistance genes with noncanonical N-terminal domains in wild tomato species.

Nucleotide-binding and leucine-rich repeat immune receptors (NLRs) provide resistance against diverse pathogens. To create comparative NLR resources, we conducted resistance gene enrichment sequencing (RenSeq) with single-molecule real-time sequencing of PacBio for 18 accessions in Solanaceae, including 15 accessions of five wild tomato species. We investigated the evolution of a class of NLRs, CNLs with extended N-terminal sequences previously named Solanaceae Domain. Through comparative genomic analysis, we revealed that the extended CNLs (exCNLs) anciently emerged in the most recent common ancestor between Asterids and Amaranthaceae, far predating the Solanaceae family. In tomatoes, the exCNLs display exceptional modes of evolution in a clade-specific manner. In the clade G3, exCNLs have substantially elongated their N-termini through tandem duplications of exon segments. In the clade G1, exCNLs have evolved through recent proliferation and sequence diversification. In the clade G6, an ancestral exCNL has lost its N-terminal domains in the course of evolution. Our study provides high-quality NLR gene models for close relatives of domesticated tomatoes that can serve as a useful resource for breeding and molecular engineering for disease resistance. Our findings regarding the exCNLs offer unique backgrounds and insights for future functional studies of the NLRs.

NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana.

Effector-triggered immunity (ETI) in plants involves a large family of nucleotide-binding leucine-rich repeat (NLR) immune receptors, including Toll/IL-1 receptor-NLRs (TNLs) and coiled-coil NLRs (CNLs). Although various NLR immune receptors are known, a mechanistic understanding of NLR function in ETI remains unclear. The TNL Recognition of XopQ 1 (Roq1) recognizes the effectors XopQ and HopQ1 from Xanthomonas and Pseudomonas, respectively, which activates resistance to Xanthomonas euvesicatoria and Xanthomonas gardneri in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana In this study, we found that the N. benthamiana N requirement gene 1 (NRG1), a CNL protein required for the tobacco TNL protein N-mediated resistance to tobacco mosaic virus, is also essential for immune signaling [including hypersensitive response (HR)] triggered by the TNLs Roq1 and Recognition of Peronospora parasitica 1 (RPP1), but not by the CNLs Bs2 and Rps2, suggesting that NRG1 may be a conserved key component in TNL signaling pathways. Besides EDS1, Roq1 and NRG1 are necessary for resistance to Xanthomonas and Pseudomonas in N. benthamiana NRG1 functions downstream of Roq1 and EDS1 and physically associates with EDS1 in mediating XopQ-Roq1-triggered immunity. Moreover, RNA sequencing analysis showed that XopQ-triggered gene-expression profile changes in N. benthamiana were almost entirely mediated by Roq1 and EDS1 and were largely regulated by NRG1. Overall, our study demonstrates that NRG1 is a key component that acts downstream of EDS1 to mediate various TNL signaling pathways, including Roq1 and RPP1-mediated HR, resistance to Xanthomonas and Pseudomonas, and XopQ-regulated transcriptional changes in N. benthamiana.