Rhizoviticin is an alphaproteobacterial tailocin that mediates biocontrol of grapevine crown gall disease.

Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control.

Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses.

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity responses. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2, present in the apoplast, depends on ribonuclease catalytic residues. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

Subgenome dominance shapes novel gene evolution in the decaploid pitcher plant Nepenthes gracilis.

Subgenome dominance after whole-genome duplication generates distinction in gene number and expression at the level of chromosome sets, but it remains unclear how this process may be involved in evolutionary novelty. Here we generated a chromosome-scale genome assembly of the Asian pitcher plant Nepenthes gracilis to analyse how its novel traits (dioecy and carnivorous pitcher leaves) are linked to genomic evolution. We found a decaploid karyotype and a clear indication of subgenome dominance. A male-linked and pericentromerically located region on the putative sex chromosome was identified in a recessive subgenome and was found to harbour three transcription factors involved in flower and pollen development, including a likely neofunctionalized LEAFY duplicate. Transcriptomic and syntenic analyses of carnivory-related genes suggested that the paleopolyploidization events seeded genes that subsequently formed tandem clusters in recessive subgenomes with specific expression in the digestive zone of the pitcher, where specialized cells digest prey and absorb derived nutrients. A genome-scale analysis suggested that subgenome dominance likely contributed to evolutionary innovation by permitting recessive subgenomes to diversify functions of novel tissue-specific duplicates. Our results provide insight into how polyploidy can give rise to novel traits in divergent and successful high-ploidy lineages.

Apoplastic Expression of CARD1-ecto Domain in Nicotiana benthamiana and Purification from the Apoplastic Fluids.

The protein expression and purification process is an essential initial step for biochemical analysis of a protein of interest. Traditionally, heterologous protein expression systems (such as E. coli, yeast, insect cells, and cell-free) are employed for plant protein expression, although a plant expression system is often desirable for plant proteins, to ensure proper post-translational modifications. Here, we describe a method to express and purify the ectodomain of one of the leucine-rich repeat receptor-like kinase called CARD1/HPCA1, from Nicotiana benthamiana apoplastic fluid. First, we express His-tagged CARD1 ectodomain in the apoplastic space of N. benthamiana by the Agroinfiltration method. Then, we collect apoplastic fluids from the leaves and purify the His-tagged protein by Ni2+-affinity chromatography. In addition to plant-specific post-translational modifications, protein accumulated in the plant apoplastic space, rather than in the cytosolic space, should be kept under an oxidizing environment. Such an environment will help to maintain the property of intrinsic disulfide bonds in the protein of interest. Further, purification from the apoplastic fluids, rather than the total protein extract, will significantly reduce contaminants (for instance RuBisCO) during protein extraction, and simplify downstream processes. We envisage that our system will be useful for expressing various plant proteins, particularly the apoplastic or extracellular regions of membrane proteins.

Interfamily grafting capacity of petunia.

In grafting, an agricultural technique for propagating flower species and fruit trees, two plants are combined to exploit their beneficial characteristics, such as rootstock disease tolerance and vigor. Grafting incompatibility has been observed, however, between distantly related plant combinations, which limits the availability of plant resources. A high grafting capacity has been found in Nicotiana, belonging to Solanaceae, but not in Ipomoea nil, a Convolvulaceae species. Here, we found that Petunia hybrida, another solanaceous species, has similar ability of interfamily grafting, which indicates that interfamily grafting capability in Solanaceae is not limited to the genus Nicotiana. RNA sequencing-based comparative time-series transcriptomic analyses of Nicotiana benthamiana, I. nil, and P. hybrida revealed that N. benthamiana and P. hybrida share a common gene expression pattern, with continued elevated expression of the ß-1,4-glucanase subclade gene GH9B3 observed after interfamily grafting. During self-grafting, GH9B3 expression in each species was similarly elevated, thus suggesting that solanaceous plants have altered regulatory mechanisms for GH9B3 gene expression that allow tissue fusion even with other species. Finally, we tested the effect of the ß-1,4-glucanase inhibitor D-glucono-1,5-lactone, using glucose as a control, on the interfamily grafting usability of P. hybrida with Arabidopsis rootstock. Strong inhibition of graft establishment was observed only with D-glucono-1,5-lactone, thus suggesting the important role of GH9B3 in P. hybrida grafting. The newly discovered grafting compatibility of Petunia with different families enhances the propagation techniques and the production of flower plants.

Oxicam-type non-steroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway.

Nonsteroidal anti-inflammatory drugs (NSAIDs), including salicylic acid (SA), target mammalian cyclooxygenases. In plants, SA is a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identify that the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent plant immune response. TNX treatment decreases NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. A cysteine labeling assay reveals that cysteine residues in NPR1 can be oxidized in vitro, leading to disulfide-bridged oligomerization of NPR1, but not in vivo regardless of SA or TNX treatment. Therefore, this study indicates that oxicam inhibits NPR1-mediated SA signaling without affecting the redox status of NPR1.

Two novel poty-like viruses identified from the transcriptome data of purple witchweed (Striga hermonthica).

Potyvirids (the family Potyviridae) are the largest family of plant RNA viruses. Two novel potyvirid viruses, Striga-associated poty-like virus 1 (SaPlV1) and Striga-associated poty-like virus 2 (SaPlV2), were identified from the transcriptome data of purple witchweed (Striga hermonthica). SaPlV1 was most closely related to bellflower veinal mottle virus (BVMoV), the only member of the genus Bevemovirus, and then to macluraviruses (the genus Macluravirus). The SaPlV1 genome encodes a 2462-amino acid (aa) polyprotein that may be cleaved into nine mature peptides. The cleavage sites of SaPlV1, BVMoV, and macluravirus polyproteins shared strong sequence similarities. SaPlV2 was most closely related to celery latent virus, the sole species of the genus Celavirus, which is the most divergent potyvirid genus. The SaPlV2 polyprotein contained 3329 aa and it may be cleaved into at least seven or eight mature peptides. Phylogenetic analysis suggested that SaPlV1 and SaPlV2 may be novel species of the genera Bevemovirus and Celavirus, respectively. The genome sequences of SaPlV1 and SaPlV2 are useful resources for studying the genome evolution of potyvirids. Keywords: Striga-associated poty-like virus 1; Striga-associated poty-like virus 2; Potyviridae; Beve- movirus; Celavirus; purple witchweed; Striga hermonthica.

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.

The Conserved Colletotrichum spp. Effector Candidate CEC3 Induces Nuclear Expansion and Cell Death in Plants.

Plant pathogens secrete proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these putative conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.

Complete Genome Sequence of Pseudomonas amygdali pv. tabaci Strain 6605, a Causal Agent of Tobacco Wildfire Disease.

Pseudomonas amygdali pv. tabaci strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer.

Comparative transient expression analyses on two conserved effectors of Colletotrichum orbiculare reveal their distinct cell death-inducing activities between Nicotiana benthamiana and melon.

Colletotrichum orbiculare infects cucurbits, such as cucumber and melon (Cucumis melo), as well as the model Solanaceae plant Nicotiana benthamiana, by secreting an arsenal of effectors that suppress the immunity of these distinct plants. Two conserved effectors of C. orbiculare, called NLP1 and NIS1, induce cell death responses in N. benthamiana, but it is unclear whether they exhibit the same activity in Cucurbitaceae plants. In this study, we established a new Agrobacterium-mediated transient expression system to investigate the cell death-inducing activity of NLP1 and NIS1 in melon. NLP1 strongly induced cell death in melon but, in contrast to the effects seen in N. benthamiana, mutations either in the heptapeptide motif or in the putative glycosylinositol phosphorylceramide-binding site did not cancel its cell death-inducing activity in melon. Furthermore, NLP1 lacking the signal peptide caused cell death in melon but not in N. benthamiana. Study of the transient expression of NIS1 also revealed that, unlike in N. benthamiana, NIS1 did not induce cell death in melon. In contrast, NIS1 suppressed flg22-induced reactive oxygen species generation in melon, as seen in N. benthamiana. These findings indicate distinct cell death-inducing activities of NLP1 and NIS1 in these two plant species that C. orbiculare infects.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.

Ethylene signaling mediates host invasion by parasitic plants.

Parasitic plants form a specialized organ, a haustorium, to invade host tissues and acquire water and nutrients. To understand the molecular mechanism of haustorium development, we performed a forward genetics screening to isolate mutants exhibiting haustorial defects in the model parasitic plant Phtheirospermum japonicum. We isolated two mutants that show prolonged and sometimes aberrant meristematic activity in the haustorium apex, resulting in severe defects on host invasion. Whole-genome sequencing revealed that the two mutants respectively have point mutations in homologs of ETHYLENE RESPONSE 1 (ETR1) and ETHYLENE INSENSITIVE 2 (EIN2), signaling components in response to the gaseous phytohormone ethylene. Application of the ethylene signaling inhibitors also caused similar haustorial defects, indicating that ethylene signaling regulates cell proliferation and differentiation of parasite cells. Genetic disruption of host ethylene production also perturbs parasite invasion. We propose that parasitic plants use ethylene as a signal to invade host roots.

Quinone perception in plants via leucine-rich-repeat receptor-like kinases.

Quinones are produced and sensed in all kingdoms of life1-4. Plants are primary producers of quinone1,2, but the role of quinone as a signalling agent in plants remains largely unknown. One well-documented role of quinone is in the induction of haustoria (specialized feeding structures) in plants that parasitize roots, which occurs in the presence of the host-derived quinone compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ)5. However, how parasitic plants sense DMBQ remains unclear, as is whether nonparasitic plants are capable of sensing quinones. Here we use Arabidopsis thaliana and DMBQ as a model plant and quinone to show that DMBQ signalling occurs in Arabidopsis via elevation of cytosolic Ca2+ concentration. We performed a forward genetic screen in Arabidopsis that isolated DMBQ-unresponsive mutants, which we named cannot respond to DMBQ 1 (card1). The CANNOT RESPOND TO DMBQ 1 (CARD1; At5g49760, also known as HPCA1) gene encodes a leucine-rich-repeat receptor-like kinase that is highly conserved in land plants. In Arabidopsis, DMBQ triggers defence-related gene expression, and card1 mutants show impaired immunity against bacterial pathogens. In Phtheirospermum japonicum (a plant that parasitizes roots), DMBQ initiates Ca2+ signalling in the root and is important for the development of the haustorium. Furthermore, CARD1 homologues from this parasitic plant complement DMBQ-induced elevation of cytosolic Ca2+ concentration in the card1 mutant. Our results demonstrate that plants-unlike animals and bacteria-use leucine-rich-repeat receptor-like kinases for quinone signalling. This work provides insights into the role of quinone signalling and CARD1 functions in plants that help us to better understand the signalling pathways used during the formation of the haustorium in parasitic plants and in plant immunity in nonparasitic plants.

Cell-cell adhesion in plant grafting is facilitated by ß-1,4-glucanases.

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of ß-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the ß-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.

Host-parasite tissue adhesion by a secreted type of ß-1,4-glucanase in the parasitic plant Phtheirospermum japonicum.

Tissue adhesion between plant species occurs both naturally and artificially. Parasitic plants establish intimate relationship with host plants by adhering tissues at roots or stems. Plant grafting, on the other hand, is a widely used technique in agriculture to adhere tissues of two stems. Here we found that the model Orobanchaceae parasitic plant Phtheirospermum japonicum can be grafted on to interfamily species. To understand molecular basis of tissue adhesion between distant plant species, we conducted comparative transcriptome analyses on both infection and grafting by P. japonicum on Arabidopsis. Despite different organs, we identified the shared gene expression profile, where cell proliferation- and cell wall modification-related genes are up-regulated. Among genes commonly induced in tissue adhesion between distant species, we showed a gene encoding a secreted type of ß-1,4-glucanase plays an important role for plant parasitism. Our data provide insights into the molecular commonality between parasitism and grafting in plants.

An auxin transport network underlies xylem bridge formation between the hemi-parasitic plant Phtheirospermum japonicum and host Arabidopsis.

Parasitic plants form vascular connections with host plants for efficient material transport. The haustorium is the responsible organ for host invasion and subsequent vascular connection. After invasion of host tissues, vascular meristem-like cells emerge in the central region of the haustorium, differentiate into tracheary elements and establish a connection, known as a xylem bridge, between parasite and host xylem systems. Despite the importance of this parasitic connection, the regulatory mechanisms of xylem bridge formation are unknown. Here, we show the role of auxin and auxin transporters during the process of xylem bridge formation using an Orobanchaceae hemiparasitic plant, Phtheirospermum japonicum The auxin response marker DR5 has a similar expression pattern to tracheary element differentiation genes in haustoria. Auxin transport inhibitors alter tracheary element differentiation in haustoria, but biosynthesis inhibitors do not, demonstrating the importance of auxin transport during xylem bridge formation. The expression patterns and subcellular localization of PIN family auxin efflux carriers and AUX1/LAX influx carriers correlate with DR5 expression patterns. The cooperative action of auxin transporters is therefore responsible for controlling xylem vessel connections between parasite and host.

An artificial metalloenzyme biosensor can detect ethylene gas in fruits and Arabidopsis leaves.

Enzyme biosensors are useful tools that can monitor rapid changes in metabolite levels in real-time. However, current approaches are largely constrained to metabolites within a limited chemical space. With the rising development of artificial metalloenzymes (ArM), a unique opportunity exists to design biosensors from the ground-up for metabolites that are difficult to detect using current technologies. Here we present the design and development of the ArM ethylene probe (AEP), where an albumin scaffold is used to solubilize and protect a quenched ruthenium catalyst. In the presence of the phytohormone ethylene, cross metathesis can occur to produce fluorescence. The probe can be used to detect both exogenous- and endogenous-induced changes to ethylene biosynthesis in fruits and leaves. Overall, this work represents an example of an ArM biosensor, designed specifically for the spatial and temporal detection of a biological metabolite previously not accessible using enzyme biosensors.

Plant Immune Responses to Parasitic Nematodes.

Plant-parasitic nematodes (PPNs), such as root-knot nematodes (RKNs) and cyst nematodes (CNs), are among the most devastating pests in agriculture. RKNs and CNs induce redifferentiation of root cells into feeding cells, which provide water and nutrients to these nematodes. Plants trigger immune responses to PPN infection by recognizing PPN invasion through several different but complementary systems. Plants recognize pathogen-associated molecular patterns (PAMPs) sderived from PPNs by cell surface-localized pattern recognition receptors (PRRs), leading to pattern-triggered immunity (PTI). Plants can also recognize tissue and cellular damage caused by invasion or migration of PPNs through PRR-based recognition of damage-associated molecular patterns (DAMPs). Resistant plants have the added ability to recognize PPN effectors via intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type immune receptors, leading to NLR-triggered immunity. Some PRRs may also recognize apoplastic PPN effectors and induce PTI. Plant immune responses against PPNs include the secretion of anti-nematode enzymes, the production of anti-nematode compounds, cell wall reinforcement, production of reactive oxygen species and nitric oxide, and hypersensitive response-mediated cell death. In this review, we summarize the recognition mechanisms for PPN infection and what is known about PPN-induced immune responses in plants.

Publisher Correction: A downy mildew effector evades recognition by polymorphism of expression and subcellular localization.

The original version of this article contained an error in the author affiliations. Oliver J. Furzer was incorrectly associated with Department of Plant Sciences, College of Life Sciences, Wuhan University, 430072, Wuhan, China.This has now been corrected in the HTML version of the article. The PDF version of the article was correct at the time of publication.Furthermore, the original version of this article stated that correspondence and requests for materials should be addressed to Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany S.A. (email: shuta.asai@riken.jp) or to J.D.G.J. (email: jonathan.jones@tsl.ac.uk). The words "Heidelberg.Center.for.Personalized.Oncology, DKFZ-HIPO, DKFZ, Heidelberg 69120Germany" were introduced inadvertently.This has now been corrected in the PDF version of the article. The HTML version of the article was correct at the time of publication.