XopQ induced stromule formation in Nicotiana benthamiana is causally linked to ETI signaling and depends on ADR1 and NRG1

In Nicotiana benthamiana, expression of the Xanthomonas effector XopQ triggers ROQ1-dependent ETI responses and in parallel accumulation of plastids around the nucleus and the formation of stromules. Both processes were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here we utilized transient expression experiments to determine whether XopQ-mediated plastid reactions are a result of XopQ perception by ROQ1 or a consequence of XopQ virulence activity. We find that N. benthamiana mutants lacking ROQ1, both RNLs (NRG1 and ADR1) or EDS1, fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the RNL double mutant. This analysis aligns XopQ-induced stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast peri-nuclear dynamics, is an integral part of the N. benthamiana ETI response and that both RNL sub-types play a role in this ETI response.

AP2/ERF transcription factor NbERF-IX-33 is involved in the regulation of phytoalexin production for the resistance of Nicotiana benthamiana to Phytophthora infestans.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated for capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies) and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair

Cooperation between receptors from the NLR superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

Identification of Micro Ribonucleic Acids and Their Targets in Response to Plasmodiophora brassicae Infection in Brassica napus

Clubroot disease, which is caused by the soil-borne pathogen Plasmodiophora brassicae War (P. brassicae), is one of the oldest and most destructive diseases of Brassica and cruciferous crops in the world. Plant microRNAs [micro ribonucleic acids (miRNAs)] play important regulatory roles in several developmental processes. Although the role of plant miRNAs in plant-microbe interaction has been extensively studied, there are only few reports on the specific functions of miRNAs in response to P. brassicae. This study investigated the roles of miRNAs and their targets during P. brassicae infection in a pair of Brassica napus near-isogenic lines (NILs), namely clubroot-resistant line 409R and clubroot-susceptible line 409S. Small RNA sequencing (sRNA-seq) and degradome-seq were performed on root samples of 409R and 409S with or without P. brassicae inoculation. sRNA-seq identified a total of 48 conserved and 72 novel miRNAs, among which 18 had a significant differential expression in the root of 409R, while only one miRNA was differentially expressed in the root of 409S after P. brassicae inoculation. The degradome-seq analysis identified 938 miRNA target transcripts, which are transcription factors, enzymes, and proteins involved in multiple biological processes and most significantly enriched in the plant hormone signal transduction pathway. Between 409R and 409S, we found eight different degradation pathways in response to P. brassicae infection, such as those related to fatty acids. By combining published transcriptome data, we identified a total of six antagonistic miRNA-target pairs in 409R that are responsive to P. brassicae infection and involved in pathways associated with root development, hypersensitive cell death, and chloroplast metabolic synthesis. Our results reveal that P. brassicae infection leads to great changes in miRNA pool and target transcripts. More interestingly, these changes are different between 409R and 409S. Clarification of the crosstalk between miRNAs and their targets may shed new light on the possible mechanisms underlying the pathogen resistance against P. brassicae.

The helper NLR immune protein NRC3 mediates the hypersensitive cell death caused by the cell-surface receptor Cf-4

Cell surface pattern recognition receptors (PRRs) activate immune responses that can include the hypersensitive cell death. However, the pathways that link PRRs to the cell death response are poorly understood. Here, we show that the cell surface receptor-like protein Cf-4 requires the intracellular nucleotide-binding domain leucine-rich repeat containing receptor (NLR) NRC3 to trigger a confluent cell death response upon detection of the fungal effector Avr4 in leaves of Nicotiana benthamiana. This NRC3 activity requires an intact N-terminal MADA motif, a conserved signature of coiled-coil (CC)-type plant NLRs that is required for resistosome-mediated immune responses. A chimeric protein with the N-terminal α1 helix of Arabidopsis ZAR1 swapped into NRC3 retains the capacity to mediate Cf-4 hypersensitive cell death. Pathogen effectors acting as suppressors of NRC3 can suppress Cf-4-triggered hypersensitive cell-death. Our findings link the NLR resistosome model to the hypersensitive cell death caused by a cell surface PRR.

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.

Genome-wide identification of PTI1 family in Setaria italica and salinity-responsive functional analysis of SiPTI1–5

Background PTI1 (Pto-interacting 1) protein kinase belongs to the receptor-like cytoplasmic kinase (RLCK) group of receptor-like protein kinases (RLK), but lack extracellular and transmembrane domains. PTI1 was first identified in tomato (Solanum lycopersicum) and named SlPTI1, which has been reported to interact with bacterial effector Pto, a serine/threonine protein kinase involved in plant resistance to bacterial disease. Briefly, the host PTI1 specifically recognizes and interacts with the bacterial effector AvrPto, which triggers hypersensitive cell death to inhibit the pathogen growth in the local infection site. Previous studies have demonstrated that PTI1 is associated with oxidative stress and hypersensitivity. Results We identified 12 putative PTI1 genes from the genome of foxtail millet (Setaria italica) in this study. Gene replication analysis indicated that both segmental replication events played an important role in the expansion of PTI1 gene family in foxtail millet. The PTI1 family members of model plants, i.e. S. italica, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), maize (Zea mays), S. lycopersicum, and soybean (Glycine max), were classified into six major categories according to the phylogenetic analysis, among which the PTI1 family members in foxtail millet showed higher degree of homology with those of rice and maize. The analysis of a complete set of SiPTI1 genes/proteins including classification, chromosomal location, orthologous relationships and duplication. The tissue expression characteristics revealed that SiPTI1 genes are mainly expressed in stems and leaves. Experimental qRT-PCR results demonstrated that 12 SiPTI1 genes were induced by multiple stresses. Subcellular localization visualized that all of foxtail millet SiPTI1s were localized to the plasma membrane. Additionally, heterologous expression of SiPTI1–5 in yeast and E. coli enhanced their tolerance to salt stress. Conclusions Our results contribute to a more comprehensive understanding of the roles of PTI1 protein kinases and will be useful in prioritizing particular PTI1 for future functional validation studies in foxtail millet.

Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair

Cooperation between receptors from the NLR superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

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

ABSTRACTIn plants, NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NRCs (NLR required for cell death), 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 hypothesized that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 167 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 five of the identified suppressors, one cyst nematode protein and one 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.

A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted Gram-negative bacterium that is the causal agent of citrus huanglongbing (HLB). In this study, we identified a CLas-encoded Sec-dependent secretory protein CLIBASIA_04405 that could contribute to the pathogenicity of this bacterium. The gene expression level of CLIBASIA_04405 was significantly higher in citrus than in psyllids. Transient overexpression of the mature CLIBASIA_04405 protein (m4405) in Nicotiana benthamiana leaves could suppress hypersensitive response (HR)-based cell death and H2O2 accumulation triggered by the mouse BAX and the Phytophthora infestans INF1. An alanine-substitution mutagenesis assay revealed the essential of amino acid clusters EKR45–47 and DE64–65 in cell death suppression. Challenge inoculation of the transgenic N. benthamiana-expressing m4405 with Pseudomonas syringae DC3000ΔhopQ1-1 demonstrated the greatly reduced bacterial proliferation. Remarkably, transcriptome profiling and RT-qPCR analysis disclosed that the gene expression of six small heat shock proteins (sHSPs), a set of plant defense regulators, were significantly elevated in the transgenic m4405 lines compared with those in wild-type N. benthamiana. In addition, the transgenic m4405 lines displayed phenotypes of dwarfism and leaf deformation. Altogether, these data indicated that m4405 was a virulence factor of CLas.