Whole Genome Resources of 17 Curtobacterium flaccumfaciens Strains Including Pathotypes of C. flaccumfaciens pv. betae, C. flaccumfaciens pv. oortii and C. flaccumfaciens pv. poinsettiae

Curtobacterium flaccumfaciens complex species in the family Microbacteriaceae encompasses a group of plant pathogenic actinobacterial strains affecting annual crops and ornamental plants. The species includes five pathovars namely C. flaccumfaciens pv. betae, C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. ilicis, C. flaccumfaciens pv. oortii, and C. flaccumfaciens pv. poinsettiae. Despite the economic importance of C. flaccumfaciens, its members have rarely been investigated for their phylogenetic relationships, molecular characteristics and virulence repertories due in part to the lack of whole genome resources. Here we present the whole genome sequence of 17 C. flaccumfaciens strains representing members of four pathovars isolated from different plant species in a diverse geographical and temporal span. The genomic data presented in this study will pave the way of research on the comparative genomics, phylogenomics and taxonomy of C. flaccumfaciens, and extend our understanding of the virulence features of the species.

Proteomics and lipidomics integrated analysis of outer membrane vesicles indicates that glycerophospholipid metabolism contributes to the early symbiosis between Sinorhizobium fredii HH103 and soybean

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited so far. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that was set as control (C-OMVs) and symbiosis-mimicking conditions that was induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism and peptidoglycan biosynthesis. A total of 45 differential lipids were identified in lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched KEGG metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by ELISA. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.

Bradyrhizobium japonicum IRAT FA3 alters Arabidopsis thaliana root architecture via regulation of auxin efflux transporters PIN2, PIN3, PIN7 and ABCB19

Beneficial rhizobacteria can stimulate changes in plant root development. While root system growth is mediated by multiple factors, the regulated distribution of the phytohormone auxin within root tissues plays a principal role. Auxin transport facilitators help to generate the auxin gradients and maxima that determine root structure. Here, we show that the plant growth-promoting rhizobacterial strain Bradyrhizobium japonicum IRAT FA3 influences specific auxin efflux transporters to alter Arabidopsis thaliana root morphology. Gene expression profiling of host transcripts in control and B. japonicum-inoculated roots of the wild type A. thaliana accession Col-0 confirmed upregulation of PIN2, PIN3, PIN7 and ABCB19 with B. japonicum and identified genes potentially contributing to a diverse array of auxin-related responses. Co-cultivation of the bacterium with loss-of-function auxin efflux transport mutants revealed that B. japonicum requires PIN3, PIN7 and ABCB19 to increase lateral root development and utilizes PIN2 to reduce primary root length. Accelerated lateral root primordia production due to B. japonicum was not observed in single pin3, pin7 or abcb19 mutants, suggesting independent roles for PIN3, PIN7 and ABCB19 during the plant-microbe interaction. Our work demonstrates B. japonicum’s influence over host transcriptional reprogramming during plant interaction with this beneficial microbe and the subsequent alterations to root system architecture.

Colonization of mutualistic mycorrhizal and parasitic blast fungi requires OsRAM2-regulated fatty acid biosynthesis in rice

Arbuscular mycorrhizal (AM) fungi form a mutual association with the majority of land plants, including most angiosperms of the dicotyledon and monocotyledon lineages. The symbiosis is based upon bidirectional nutrient exchange between the host and symbiont that occurs between inner cortical cells of the root and branched AM hyphae called arbuscules that develop within these cells. Lipid transport and its regulation during the symbiosis have been intensively investigated in dicotyledon plants, especially legumes. Here, we characterize OsRAM2 and OsRAM2L, homologs of M. truncatula RAM2, and found that plants defective in OsRAM2 were unable to be colonized by AM fungi and showed impaired colonization by Magnaporthe oryzae. The induction of OsRAM2 and OsRAM2L is dependent on OsRAM1 and the CSSP pathway genes CCaMK and CYCLOPS, while overexpression of OsRAM1 results in increased expression of OsRAM2 and OsRAM2L. Collectively, our data show that the function and regulation of OsRAM2 is conserved in monocot and dicot plants and reveals that, similar to mutualistic fungi, pathogenic fungi have recruited RAM2-mediated fatty acid biosynthesis to facilitate invasion.

‘Candidatus Liberibacter asiaticus’-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress in Planta

The lipopolysaccharides (LPS) of Gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid (VLCFA) modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on non-host tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host citrus (Citrus sinensis, confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione (GSH) pool, callose deposition and activation of the salicylic acid (SA) and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ BCP peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (a) attenuates NO-mediated SAR signaling and (b) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.

A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus. [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 .

SGT1 SGS-domain mutations impair interactions with the barley MLA6 immune receptor in association with loss of NLR protein

The Mla (Mildew resistance locus a) of barley (Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich-repeat (CC-NLR) receptor require the HRS complex to function, which includes HSP90 (Heat Shock Protein 90), RAR1 (Required for Mla12 Resistance 1), and SGT1 (Suppressor of G-two allele of Skp1). However, functional analysis of Sgt1 has been particularly difficult as deletions are often lethal. Recently, we identified rar3 (Required for Mla6 resistance 3), an in-frame Sgt1ΔKL308-309 mutation in the SGS domain that alters resistance conferred by MLA, but without lethality. Here we use autoactive MLA6 and heterologous yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90, to determine that this mutation weakens, but doesn’t entirely disrupt, the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions.

Cross-Kingdom Gene Coexpression Analysis Using a Stemphylium botryosum–Lens ervoides System Revealed Plasticity of Intercommunication Between the Pathogen Secretome and the Host Immune Systems

Necrotrophic pathogens are responsible for significant declines in crop yield and quality worldwide. During the infection process, a pathogen releases a series of secretory proteins to counteract the plant immune system, and this interaction of pathogen and host molecules determines whether the pathogen will successfully invade the host plant tissues. In this study, we adopted co-transcriptomic approaches to analyze the Lens ervoides–Stemphylium botryosum system, with a focus on 1,216 fungal genes coding for secretory proteins and 8,810 disease-responsive genes of the host 48, 96, and 144 h postinoculation, captured in two F9 recombinant inbred lines (RILs) displaying contrasting disease responses. By constructing in planta gene coexpression networks (GCNs) for S. botryosum, we found that the pathogen tended to co-upregulate genes regulating cell wall degradation enzymes, effectors, oxidoreductases, and peptidases to a much higher degree in the susceptible host LR-66-577 than in the resistant RIL LR-66-637, indicating that the promotion of these digestive enzymes and toxins increased S. botryosum virulence. Construction of cross-kingdom GCNs between pathogen and plant for the two RILs revealed that the co-upregulation of these fungal digestive enzymes and toxins simultaneously promoted a series of defense responses such as redox change, expression of membrane-related genes and serine/threonine kinase, and stress and disease responses in the susceptible RIL which was not observed in the resistant RIL, indicating that these activities exacerbated susceptibility to S. botryosum. [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 .

Fusaricidin Biosynthesis Is Controlled via a KinB-Spo0A-AbrB Signal Pathway in Paenibacillus polymyxa WLY78

Fusaricidins produced by Paenibacillus polymyxa are important lipopeptide antibiotics against fungi. The fusGFEDCBA (fusaricidin biosynthesis) operon is responsible for synthesis of fusaricidins. However, the regulation mechanisms of fusaricidin biosynthesis remain to be fully clarified. In this study, we revealed that fusaricidin production is controlled by a complex regulatory network including KinB-Spo0A-AbrB. Evidence suggested that the regulator AbrB represses the transcription of the fus gene cluster by direct binding to the fus promoter, in which the sequences (5′-AATTTTAAAATAAATTTTGTGATTT-3′) located from −136 to −112 bp relative to the transcription start site is required for this repression. Spo0A binds to the abrB promoter that contains the Spo0A-binding sequences (5′-TGTCGAA-3′, 0A box) and in turn prevents the further transcription of abrB. The decreasing concentration of AbrB allows for the derepression of the fus promoter repressed by AbrB. The genome of P. polymyxa WLY78 contains two orthologs (named Kin1508 and Kin4833) of Bacillus subtilis KinB, but only Kin4833 activates sporulation and fusaricidin production, indicating that this kinase may be involved in phosphorylating Spo0A to initiate sporulation and regulate the abrB transcription. Our results reveal that Kin4833 (KinB), Spo0A, and AbrB are involved in regulation of fusaricidin production and a signaling mechanism that links fusaricidin production and sporulation. [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 .

Impact of future elevated carbon dioxide on C3 plant resistance to biotic stresses

Before the end of the century, atmospheric carbon dioxide (CO2) levels are predicted to increase to ~900 ppm. This will dramatically affect plant physiology and influence environmental interactions and, in particular, plant resistance to biotic stresses. This review is a broad survey of the current research on the effects of elevated CO2 (eCO2) on phytohormone-mediated resistance of C3 agricultural crops and related model species to pathogens and insect herbivores. In general, while plants grown in eCO2 often have increased constitutive and induced salicylic acid levels and suppressed induced jasmonate levels, there are exceptions that implicate other environmental factors, such as light and nitrogen fertilization in modulating these responses. Therefore, this review sets the stage for future studies to delve into understanding the mechanistic basis behind how eCO2 will affect plant defensive phytohormone signaling pathways under future predicted environmental conditions that could threaten global food security to inform the best agricultural management practices.