Revealing the secrets beneath grapevine and Plasmopara viticola early communication: a picture of host and pathogen proteomes

Plant apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors and several signal transduction pathways are activated. As a result of this first contact, the host triggers a defence response that involves the modulation of several extra and intracellular proteins. In grapevine-pathogen interactions, little is known about the communication between cells and apoplast. Also, the role of apoplastic proteins in response to pathogens still remains a blackbox. In this study we focused on the first 6 hours after Plasmopara viticola inoculation to evaluate grapevine proteome modulation in the apoplastic fluid (APF) and whole leaf tissue. Plasmopara viticola proteome was also assessed enabling a deeper understanding of plant and pathogen communication. Our results showed that oomycete recognition, plant cell wall modifications, ROS signalling and disruption of oomycete structures are triggered in Regent after P. viticola inoculation. Our results highlight a strict relation between the apoplastic pathways modulated and the proteins identified in the whole leaf proteome. On the other hand, P. viticola proteins related to growth/morphogenesis and virulence mechanisms were the most predominant. This pioneer study highlights the early dynamics of extra and intracellular communication in grapevine defence activation that leads to the successful establishment of an incompatible interaction.

The class I TCP transcription factor AtTCP8 is a modulator of phytohormone-responsive signaling networks

The plant-specific TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR (TCP) transcription factor family is most closely associated with regulating plant developmental programs. Recently, TCPs were also shown to mediate host immune signaling, both as targets of pathogen virulence factors and regulators of plant defense genes. However, any comprehensive characterization of TCP gene targets is still lacking. Loss of the class I TCP AtTCP8 attenuates early immune signaling, and when combined with mutations in AtTCP14 and AtTCP15, additional layers of defense signaling in Arabidopsis thaliana. Here we focus on TCP8, the most poorly characterized of the three to date. We use chIP and RNA-sequencing to identify TCP8-bound gene promoters and differentially regulated genes in the tcp8 mutant, data sets that are heavily enriched in signaling components for multiple phytohormone pathways, including brassinosteroids (BRs), auxin, and jasmonic acid (JA). Using BR signaling as a representative example, we show that TCP8 directly binds and activates the promoters of the key BR transcriptional regulators BZR1 and BZR2/BES1. Furthermore, tcp8 mutant seedlings exhibit altered BR-responsive growth patterns and complementary reductions in BZR2 transcript levels, while the expressed protein demonstrates BR-responsive changes in subnuclear localization and transcriptional activity. We conclude that one explanation for the significant targeting of TCP8 alongside other TCP family members by pathogen effectors may lie in its role as a modulator of brassinosteroid and other plant hormone signaling pathways.

The rice OsERF101 transcription factor regulates the NLR Xa1-mediated perception of TAL effectors and Xa1-mediated immunity

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses and the hypersensitive response by recognizing pathogen effectors. Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo). The molecular mechanisms controlling the recognition of TAL effectors by Xa1 and the subsequent induction of immunity remain poorly understood. Xa1 interacts in the nucleus with two TAL effectors via the BED domain. We identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6 as an interactor with Xa1, and found that it also interacts with the TAL effectors. Overexpression of OsERF101 exhibited an enhanced resistance to an incompatible Xoo strain only in the presence of Xa1, indicating that OsERF101 functions as a positive regulator of Xa1-mediated immunity. Unexpectedly, oserf101 mutants also showed enhanced Xa1-dependent resistance, but in a different manner from the overexpressing plants. This result revealed an additional Xa1-mediated immune pathway that is negatively regulated by OsERF101. Furthermore, OsERF101 directly interacted with the TAL effectors. Our results show that OsERF101 regulates the recognition of TAL effectors and the Xa1-mediated activation of the immune response. These data provide new insights into the molecular mechanism of NLR-mediated immunity in plants.

Comprehensive Time-Series Analysis of the Gene Expression Profile in a Susceptible Cultivar of Tree Tomato (Solanum betaceum) During the Infection of Phytophthora betacei

Solanum betaceum is a tree from the Andean region bearing edible fruits, considered an exotic export. Although there has been renewed interest in its commercialization, sustainability, and disease management have been limiting factors. Phytophthora betacei is a recently described species that causes late blight in S. betaceum. There is no general study of the response of S. betaceum, particularly, in the changes in expression of pathogenesis-related genes. In this manuscript we present a comprehensive RNA-seq time-series study of the plant response to the infection of P. betacei. Following six time points of infection, the differentially expressed genes (DEGs) involved in the defense by the plant were contextualized in a sequential manner. We documented 5,628 DEGs across all time-points. From 6 to 24 h post-inoculation, we highlighted DEGs involved in the recognition of the pathogen by the likely activation of pattern-triggered immunity (PTI) genes. We also describe the possible effect of the pathogen effectors in the host during the effector-triggered response. Finally, we reveal genes related to the susceptible outcome of the interaction caused by the onset of necrotrophy and the sharp transcriptional changes as a response to the pathogen. This is the first report of the transcriptome of the tree tomato in response to the newly described pathogen P. betacei.

A conserved glutamate residue in RPM1-interacting protein4 is ADP-ribosylated by Pseudomonas effector AvrRpm2 to activate RPM1-mediated response

Gram-negative bacterial plant pathogens directly inject effectors into their hosts to hijack and manipulate metabolism, eluding the frontier surveillance at the cell surface. The effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola functions as an ADP-ribosyl transferase, modifying RPM1-interacting protein4 (RIN4), leading to the activation of Arabidopsis resistance protein RPM1. We identified the ADP-ribosyl transferase activity of another bacterial effector AvrRpm2Psa from Pseudomonas syringae pv. actinidiae via infection using a Pseudomonas syringae pv. tomato strain following Agrobacterium-mediated transient expression of RIN4 in N. benthamiana. We conducted mutational analysis in combination with mass spectrometry to genetically locate the modified residue. We show that a conserved glutamate residue (E156) of AtRIN4 is the target site for AvrRpm2Psa by demonstrating the modified AtRIN4 with E156A substitution is no longer ADP-ribosylated. Accordingly, naturally occurring soybean and snap bean RIN4 homologs with no glutamate at the positions corresponding to the E156 of AtRIN4 are not ADP-ribosylated by AvrRpm2Psa. In contrast with another effector AvrB, modifications of potential phosphorylation sites including T166 in AtRIN4 affected neither ADP-ribosylation nor RPM1 activation by AvrRpm2Psa. This study suggests that separate biochemical reactions by different pathogen effectors may trigger the activation of the same resistance protein through distinct modifications of RIN4.

A genetically linked pair of NLR immune receptors show contrasting patterns of evolution

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, and is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal–associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained trans-species in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to recognize AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.Significance statementPlants have evolved sophisticated defense mechanisms to fend off pathogens. Plant nucleotide-binding leucine-rich repeat receptor (NLR) proteins play crucial roles in detecting pathogen molecules inside plant cells and mounting defense responses. Here, we identified the Pias gene from rice, which encodes the NLR pair Pias-1 “helper” and Pias-2 “sensor.” These proteins function together to detect the pathogen molecule AVR-Pias of Magnaporthe oryzae and defend against rice blast disease. Pias is allelic to the previously reported Pia gene. A comparison of Pias/Pia alleles among Oryza species showed that Pias/Pia helper is evolutionarily and functionally conserved, whereas Pias/Pia sensor shows highly dynamic evolution, with various host domains integrated into similar positions, allowing it to detect a wide variety of pathogen molecules.

NADase and now Ca2+ channel, what else to learn about plant NLRs?

Plant intracellular immune receptors known as NLR (Nucleotide-binding Leucine-rich repeat, NB-LRR) proteins confer resistance and cause cell death upon recognition of cognate effector proteins from pathogens. Plant NLRs contain a variable N-terminal domain: a Toll/interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain or an RPW8 (Resistance to Powdery Mildew 8)-like CC (CCR) domain. TIR-NLR, CC-NLR and CCR-NLR are known as TNL, CNL and RNL, respectively. TNLs and CNLs recognize pathogen effectors to activate cell death and defense responses, thus are regarded as sensor NLRs. RNLs are required downstream of TNLs to activate cell death and defense responses, thus are regarded as helper NLRs. Previous studies show that some TNLs form tetrameric resistosome as NAD+ cleaving enzymes to transduce signal, while some CNLs form pentameric resistosome with undefined biochemical function. Two recent breakthrough studies show that activated CNL and RNL function as Ca2+ channel to cause cell death and defense responses and provide a completely new insight into the downstream signaling events of CNL and TNL pathways.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

An NLR Integrated Domain toolkit to identify plant pathogen effector targets

SUMMARYPlant resistance genes (or NLR “Nod-like Receptors”) are known to contain atypical domains procuring them with a decoy capacity. Some of these integrated domains (or ID) allow the plant to lure the virulence determinants (“effectors”) of pathogens and triggering a specific NLR immune reaction.In this work, our goal was to generate a library of known IDs that could be screened with plant pathogen effectors in order to identify putative new effector virulence targets and NLR-effector pairs.We curated the IDs contained in NLRs from seven model and crop plant species. We cloned 52 IDs representing 31 distinct Pfam domains. This library was screened for interaction by yeast-two-hybrid with a set of 31 conserved Ralstonia solanacearum type III effectors. This screening and the further in planta interaction assay allowed us to identify three interactions, involving different IDs (kinase, DUF3542, WRKY) and two type III effectors (RipAE and PopP2).PopP2 was found to physically interact with ID#85, an atypical WRKY domain integrated in the GmNLR-ID85 NLR protein from Soybean. Using a imaging method in living plant cells, we showed that PopP2 associates with ID#85 in the nucleus. But unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, this newly identified soybean WRKY domain could not be acetylated by PopP2 and its atypical sequence (WRKYGKR) also probably renders it inefficient in plant immunity triggering.This ID toolkit is available for screening with other plant pathogen effectors and should prove useful to discover new effectors targets and potentially engineer new plant resistance genes.