Meloidogyne enterolobii MeMSP1 effector targets the glutathione-S-transferase phi GSTF family in Arabidopsis to manipulate host metabolism and promote nematode parasitism.

Meloidogyne enterolobii is an emerging root-knot nematode species that overcomes most of the nematode resistance genes in crops. Nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we show the MeMSP1 effector is secreted into giant cells and promotes M. enterolobii parasitism. Using co-immunoprecipitation and bimolecular fluorescent complementation assays, we identified glutathione-S-transferase phi GSTFs as host targets of the MeMSP1 effector. This protein family plays important roles in plant responses to abiotic and biotic stresses. We demonstrate that MeMSP1 interacts with all Arabidopsis GSTF. Moreover, we confirmed that the N-terminal region of AtGSTF9 is critical for its interaction, and atgstf9 mutant lines are more susceptible to root-knot nematode infection. Combined transcriptome and metabolome analyses showed that MeMSP1 affects the metabolic pathways of Arabidopsis thaliana, resulting in the accumulation of amino acids, nucleic acids, and their metabolites, and organic acids and the downregulation of flavonoids. Our study has shed light on a novel effector mechanism that targets plant metabolism, reducing the production of plant defence-related compounds while favouring the accumulation of metabolites beneficial to the nematode, and thereby promoting parasitism.

Silencing the conserved small nuclear ribonucleoprotein SmD1 target gene alters susceptibility to root-knot nematodes in plants.

Root-knot nematodes (RKNs) are among the most damaging pests of agricultural crops. Meloidogyne is an extremely polyphagous genus of nematodes that can infect thousands of plant species. A few genes for resistance (R-genes) to RKN suitable for use in crop breeding have been identified, but virulent strains and species of RKN have emerged that render these R-genes ineffective. Secretion of RKN effectors targeting plant functions mediates the reprogramming of root cells into specialized feeding cells, the giant cells, essential for RKN development and reproduction. Conserved targets among plant species define the more relevant strategies for controlling nematode infection. The EFFECTOR18 (EFF18) protein from M. incognita interacts with the spliceosomal small nuclear ribonucleoprotein D1 (SmD1) in Arabidopsis (Arabidopsis thaliana), disrupting its function in alternative splicing regulation and modulating the giant cell transcriptome. We show here that EFF18 is a conserved RKN-specific effector that targets this conserved spliceosomal SmD1 protein in Solanaceae. This interaction modulates alternative splicing events produced by tomato (Solanum lycopersicum) in response to M. incognita infection. The alteration of SmD1 expression by virus-induced gene silencing in Solanaceae affects giant cell formation and nematode development. Thus, our work defines a promising conserved SmD1 target gene to develop broad resistance for the control of Meloidogyne spp. in plants.

AUXIN RESPONSIVE FACTOR8 regulates development of the feeding site induced by root-knot nematodes in tomato.

Root-knot nematodes (RKN) from the genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression, and auxin is known to be a key player in their development. However, little is known about how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their miRNA167 regulators, were identified as robust gene-miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-ß-glucuronidase (GUS) fusions showed the up-regulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR (clustered regularly interspaced palindromic repeats) mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.

Copper microRNAs modulate the formation of giant feeding cells induced by the root knot nematode Meloidogyne incognita in Arabidopsis thaliana.

Root-knot nematodes (RKNs) are root endoparasites that induce the dedifferentiation of a few root cells and the reprogramming of their gene expression to generate giant hypermetabolic feeding cells. We identified two microRNA families, miR408 and miR398, as upregulated in Arabidopsis thaliana and Solanum lycopersicum roots infected by RKNs. In plants, the expression of these two conserved microRNA families is known to be activated by the SPL7 transcription factor in response to copper starvation. By combining functional approaches, we deciphered the network involving these microRNAs, their regulator and their targets. MIR408 expression was located within nematode-induced feeding cells like its regulator SPL7 and was regulated by copper. Moreover, infection assays with mir408 and spl7 knockout mutants or lines expressing targets rendered resistant to cleavage by miR398 demonstrated the essential role of the SPL7/MIR408/MIR398 module in the formation of giant feeding cells. Our findings reveal how perturbation of plant copper homeostasis, via the SPL7/MIR408/MIR398 module, modulates the development of nematode-induced feeding cells.

A Meloidogyne incognita C-type lectin effector targets plant catalases to promote parasitism.

Root-knot nematodes, Meloidogyne spp., secrete effectors to modulate plant immune responses and establish a parasitic relationship with host plants. However, the functions and plant targets of C-type lectin (CTL)-like effectors of Meloidogyne incognita remain unknown. Here, we characterized a CTL-like effector of M. incognita, MiCTL1a, and identified its target and role in nematode parasitism. In situ hybridization demonstrated the expression of MiCTL1 in the subventral glands; and in planta, immunolocalization showed its secretion during M. incognita parasitism. Virus-induced gene silencing of the MiCTL1 reduced the infection ability of M. incognita in Nicotiana benthamiana. The ectopic expression in Arabidopsis not only increased susceptibility to M. incognita but also promoted root growth. Yeast two-hybrid and co-immunoprecipitation assays revealed that MiCTL1a interacts with Arabidopsis catalases, which play essential roles in hydrogen peroxide homeostasis. Knockout or overexpression of catalases showed either increased or reduced susceptibility to M. incognita, respectively. Moreover, MiCTL1a not only reduced catalase activity in vitro and in planta but also modulated stress-related gene expressions in Arabidopsis. Our data suggest that MiCTL1a interacts with plant catalases and interferes with catalase activity, allowing M. incognita to establish a parasitic relationship with its host by fine-tuning responses mediated by reactive oxygen species.

The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation.

The root-knot nematode Meloidogyne incognita secretes specific effectors (MiEFF) and induces the redifferentiation of plant root cells into enlarged multinucleate feeding 'giant cells' essential for nematode development. Immunolocalizations revealed the presence of the MiEFF18 protein in the salivary glands of M. incognita juveniles. In planta, MiEFF18 localizes to the nuclei of giant cells demonstrating its secretion during plant-nematode interactions. A yeast two-hybrid approach identified the nuclear ribonucleoprotein SmD1 as a MiEFF18 partner in tomato and Arabidopsis. SmD1 is an essential component of the spliceosome, a complex involved in pre-mRNA splicing and alternative splicing. RNA-seq analyses of Arabidopsis roots ectopically expressing MiEFF18 or partially impaired in SmD1 function (smd1b mutant) revealed the contribution of the effector and its target to alternative splicing and proteome diversity. The comparison with Arabidopsis galls data showed that MiEFF18 modifies the expression of genes important for giant cell ontogenesis, indicating that MiEFF18 modulates SmD1 functions to facilitate giant cell formation. Finally, Arabidopsis smd1b mutants exhibited less susceptibility to M. incognita infection, and the giant cells formed on these mutants displayed developmental defects, suggesting that SmD1 plays an important role in the formation of giant cells and is required for successful nematode infection.

The root-knot nematode effector MiPDI1 targets a stress-associated protein (SAP) to establish disease in Solanaceae and Arabidopsis.

Large amounts of effectors are secreted by the oesophageal glands of plant-parasitic nematodes, but their molecular mode of action remains largely unknown. We characterized a Meloidogyne incognita protein disulphide isomerase (PDI)-like effector protein (MiPDI1) that facilitates nematode parasitism. In situ hybridization showed that MiPDI1 was expressed specifically in the subventral glands of M. incognita. It was significantly upregulated during parasitic stages. Immunolocalization demonstrated MiPDI1 secretion in planta during nematode migration and within the feeding cells. Host-induced silencing of the MiPDI1 gene affected the ability of the nematode to infect the host, whereas MiPDI1 expression in Arabidopsis increased susceptibility to M. incognita, providing evidence for a key role of MiPDI1 in M. incognita parasitism. Yeast two-hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assays showed that MiPDI1 interacted with a tomato stress-associated protein (SlSAP12) orthologous to the redox-regulated AtSAP12, which plays an important role in plant responses to abiotic and biotic stresses. SAP12 silencing or knocking out in Nicotiana benthamiana and Arabidopsis increased susceptibility to M. incognita. Our results suggest that MiPDI1 acts as a pathogenicity factor promoting disease by fine-tuning SAP-mediated responses at the interface of redox signalling, defence and stress acclimation in Solanaceae and Arabidopsis.

Hybridization and polyploidy enable genomic plasticity without sex in the most devastating plant-parasitic nematodes.

Root-knot nematodes (genus Meloidogyne) exhibit a diversity of reproductive modes ranging from obligatory sexual to fully asexual reproduction. Intriguingly, the most widespread and devastating species to global agriculture are those that reproduce asexually, without meiosis. To disentangle this surprising parasitic success despite the absence of sex and genetic exchanges, we have sequenced and assembled the genomes of three obligatory ameiotic and asexual Meloidogyne. We have compared them to those of relatives able to perform meiosis and sexual reproduction. We show that the genomes of ameiotic asexual Meloidogyne are large, polyploid and made of duplicated regions with a high within-species average nucleotide divergence of ~8%. Phylogenomic analysis of the genes present in these duplicated regions suggests that they originated from multiple hybridization events and are thus homoeologs. We found that up to 22% of homoeologous gene pairs were under positive selection and these genes covered a wide spectrum of predicted functional categories. To biologically assess functional divergence, we compared expression patterns of homoeologous gene pairs across developmental life stages using an RNAseq approach in the most economically important asexually-reproducing nematode. We showed that >60% of homoeologous gene pairs display diverged expression patterns. These results suggest a substantial functional impact of the genome structure. Contrasting with high within-species nuclear genome divergence, mitochondrial genome divergence between the three ameiotic asexuals was very low, signifying that these putative hybrids share a recent common maternal ancestor. Transposable elements (TE) cover a ~1.7 times higher proportion of the genomes of the ameiotic asexual Meloidogyne compared to the sexual relative and might also participate in their plasticity. The intriguing parasitic success of asexually-reproducing Meloidogyne species could be partly explained by their TE-rich composite genomes, resulting from allopolyploidization events, and promoting plasticity and functional divergence between gene copies in the absence of sex and meiosis.

Gene copy number variations as signatures of adaptive evolution in the parthenogenetic, plant-parasitic nematode Meloidogyne incognita.

Adaptation to changing environmental conditions represents a challenge to parthenogenetic organisms, and until now, how phenotypic variants are generated in clones in response to the selection pressure of their environment remains poorly known. The obligatory parthenogenetic root-knot nematode species Meloidogyne incognita has a worldwide distribution and is the most devastating plant-parasitic nematode. Despite its asexual reproduction, this species exhibits an unexpected capacity of adaptation to environmental constraints, for example, resistant hosts. Here, we used a genomewide comparative hybridization strategy to evaluate variations in gene copy numbers between genotypes of M. incognita resulting from two parallel experimental evolution assays on a susceptible vs. resistant host plant. We detected gene copy number variations (CNVs) associated with the ability of the nematodes to overcome resistance of the host plant, and this genetic variation may reflect an adaptive response to host resistance in this parthenogenetic species. The CNV distribution throughout the nematode genome is not random and suggests the occurrence of genomic regions more prone to undergo duplications and losses in response to the selection pressure of the host resistance. Furthermore, our analysis revealed an outstanding level of gene loss events in nematode genotypes that have overcome the resistance. Overall, our results support the view that gene loss could be a common class of adaptive genetic mechanism in response to a challenging new biotic environment in clonal animals.

A MIF-like effector suppresses plant immunity and facilitates nematode parasitism by interacting with plant annexins.

Plant-parasitic nematodes secrete numerous effectors to facilitate parasitism, but detailed functions of nematode effectors and their plant targets remain largely unknown. Here, we characterized four macrophage migration inhibitory factors (MIFs) in Meloidogyne incognita resembling the MIFs secreted by human and animal parasites. Transcriptional data showed MiMIFs are up-regulated in parasitism. Immunolocalization provided evidence that MiMIF proteins are secreted from the nematode hypodermis to the parasite surface, detected in plant tissues and giant cells. In planta MiMIFs RNA interference in Arabidopsis decreased infection and nematode reproduction. Transient expression of MiMIF-2 could suppress Bax- and RBP1/Gpa2-induced cell death. MiMIF-2 ectopic expression led to higher levels of Arabidopsis susceptibility, suppressed immune responses triggered by flg22, and impaired [Ca2+]cyt influx induced by H2O2. The immunoprecipitation of MiMIF-2-interacting proteins, followed by co-immunoprecipitation and bimolecular fluorescence complementation validations, revealed specific interactions between MiMIF-2 and two Arabidopsis annexins, AnnAt1 and AnnAt4, involved in the transport of calcium ions, stress responses, and signal transduction. Suppression of expression or overexpression of these annexins modified nematode infection. Our results provide functional evidence that nematode effectors secreted from hypodermis to the parasite cuticle surface target host proteins and M. incognita uses MiMIFs to promote parasitism by interfering with the annexin-mediated plant immune responses.

Characterization of siRNAs clusters in Arabidopsis thaliana galls induced by the root-knot nematode Meloidogyne incognita.

BACKGROUND: Root-knot nematodes (RKN), genus Meloidogyne, are plant parasitic worms that have the ability to transform root vascular cylinder cells into hypertrophied, multinucleate and metabolically over-active feeding cells. Redifferentiation into feeding cells is the result of a massive transcriptional reprogramming of root cells targeted by RKN. Since RKN are able to induce similar feeding cells in roots of thousands of plant species, these worms are thought to manipulate essential and conserved plant molecular pathways. RESULTS: Small non-coding RNAs of uninfected roots and infected root galls induced by M. incognita from Arabidopsis thaliana were sequenced by high throughput sequencing. SiRNA populations were analysed by using the Shortstack algorithm. We identified siRNA clusters that are differentially expressed in infected roots and evidenced an over-representation of the 23-24 nt siRNAs in infected tissue. This size corresponds to heterochromatic siRNAs (hc-siRNAs) which are known to regulate expression of transposons and genes at the transcriptional level, mainly by inducing DNA methylation. CONCLUSIONS: Correlation of siRNA clusters expression profile with transcriptomic data identified several protein coding genes that are candidates to be regulated by siRNAs at the transcriptional level by RNA directed DNA methylation (RdDM) pathway either directly or indirectly via silencing of neighbouring transposable elements.

Genome-wide expert annotation of the epigenetic machinery of the plant-parasitic nematodes Meloidogyne spp., with a focus on the asexually reproducing species.

BACKGROUND: The renewed interest in epigenetics has led to the understanding that both the environment and individual lifestyle can directly interact with the epigenome to influence its dynamics. Epigenetic phenomena are mediated by DNA methylation, stable chromatin modifications and non-coding RNA-associated gene silencing involving specific proteins called epigenetic factors. Multiple organisms, ranging from plants to yeast and mammals, have been used as model systems to study epigenetics. The interactions between parasites and their hosts are models of choice to study these mechanisms because the selective pressures are strong and the evolution is fast. The asexually reproducing root-knot nematodes (RKN) offer different advantages to study the processes and mechanisms involved in epigenetic regulation. RKN genomes sequencing and annotation have identified numerous genes, however, which of those are involved in the adaption to an environment and potentially relevant to the evolution of plant-parasitism is yet to be discovered. RESULTS: Here, we used a functional comparative annotation strategy combining orthology data, mining of curated genomics as well as protein domain databases and phylogenetic reconstructions. Overall, we show that (i) neither RKN, nor the model nematode Caenorhabditis elegans possess any DNA methyltransferases (DNMT) (ii) RKN do not possess the complete machinery for DNA methylation on the 6th position of adenine (6mA) (iii) histone (de)acetylation and (de)methylation pathways are conserved between C. elegans and RKN, and the corresponding genes are amplified in asexually reproducing RKN (iv) some specific non-coding RNA families found in plant-parasitic nematodes are dissimilar from those in C. elegans. In the asexually reproducing RKN Meloidogyne incognita, expression data from various developmental stages supported the putative role of these proteins in epigenetic regulations. CONCLUSIONS: Our results refine previous predictions on the epigenetic machinery of model species and constitute the most comprehensive description of epigenetic factors relevant to the plant-parasitic lifestyle and/or asexual mode of reproduction of RKN. Providing an atlas of epigenetic factors in RKN is an informative resource that will enable researchers to explore their potential role in adaptation of these parasites to their environment.

A root-knot nematode small glycine and cysteine-rich secreted effector, MiSGCR1, is involved in plant parasitism.

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.

Characterization of microRNAs from Arabidopsis galls highlights a role for miR159 in the plant response to the root-knot nematode Meloidogyne incognita.

Root knot nematodes (RKN) are root parasites that induce the genetic reprogramming of vascular cells into giant feeding cells and the development of root galls. MicroRNAs (miRNAs) regulate gene expression during development and plant responses to various stresses. Disruption of post-transcriptional gene silencing in Arabidopsis ago1 or ago2 mutants decrease the infection rate of RKN suggesting a role for this mechanism in the plant-nematode interaction. By sequencing small RNAs from uninfected Arabidopsis roots and from galls 7 and 14 d post infection with Meloidogyne incognita, we identified 24 miRNAs differentially expressed in gall as putative regulators of gall development. Moreover, strong activity within galls was detected for five miRNA promoters. Analyses of nematode development in an Arabidopsis miR159abc mutant had a lower susceptibility to RKN, suggesting a role for the miR159 family in the plant response to M. incognita. Localization of mature miR159 within the giant and surrounding cells suggested a role in giant cell and gall. Finally, overexpression of miR159 in galls at 14 d post inoculation was associated with the repression of the miR159 target MYB33 which expression is restricted to the early stages of infection. Overall, these results implicate the miR159 in plant responses to RKN.

The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant Cells Induced by Plant-Parasitic Nematodes in Arabidopsis.

In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKs or Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected by plant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita-induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise, phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearance of multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblance between KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in the multinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plant KRP6 transcription to the benefit of gall establishment.

Resistance to root-knot nematodes Meloidogyne spp. in woody plants.

I. 42 II. 43 III. 44 IV. 47 V. 49 VI. 50 VII. 50 VIII. 50 IX. 52 52 References 52 SUMMARY: Root-knot nematodes (RKNs) Meloidogyne spp. cause major damage to cultivated woody plants. Among them, Prunus, grapevine and coffee are the crops most infested by worldwide polyphagous species and species with a more limited distribution and/or narrower host range. The identification and characterization of natural sources of resistance are important steps to develop RKN control strategies. In woody crops, resistant rootstocks genetically different from the scion of agronomical interest may be engineered. We describe herein the interactions between RKNs and different woody crops, and highlight the plant species in which resistance and corresponding resistance (R) genes have been discovered. Even though grapevine and, to a lesser extent, coffee have a history of rootstock selection for RKN resistance, few cases of resistance have been documented. By contrast, in Prunus, R genes with different spectra have been mapped in plums, peach and almond and can be pyramided for durable resistance in interspecific rootstocks. We particularly discuss here the Ma Toll/interleukin-1 receptor-like-nucleotide binding-leucine-rich repeat gene from Myrobalan plum, one of the longest plant R genes cloned to date, due to its unique biological and structural properties. RKN R genes in Prunus will enable us to carry out molecular studies aimed at improving our knowledge of plant immunity in woody plants.

Evolutionarily distant pathogens require the Arabidopsis phytosulfokine signalling pathway to establish disease.

Secreted peptides and their specific receptors frequently orchestrate cell-to-cell communication in plants. Phytosulfokines (PSKs) are secreted tyrosine-sulphated peptide hormones, which trigger cellular dedifferentiation and redifferentiation upon binding to their membrane receptor. Biotrophic plant pathogens frequently trigger the differentiation of host cells into specialized feeding structures, which are essential for successful infection. We found that oomycete and nematode infections were characterized by the tissue-specific transcriptional regulation of genes encoding Arabidopsis PSKs and the PSK receptor 1 (PSKR1). Subcellular analysis of PSKR1 distribution showed that the plasma membrane-bound receptor internalizes after binding of PSK-α. Arabidopsis pskr1 knockout mutants were impaired in their susceptibility to downy mildew infection. Impaired disease susceptibility depends on functional salicylic acid (SA) signalling, but not on the massive up-regulation of SA-associated defence-related genes. Knockout pskr1 mutants also displayed a major impairment of root-knot nematode reproduction. In the absence of functional PSKR1, giant cells arrested their development and failed to fully differentiate. Our findings indicate that the observed restriction of PSK signalling to cells surrounding giant cells contributes to the isotropic growth and maturation of nematode feeding sites. Taken together, our data suggest that PSK signalling in Arabidopsis promotes the differentiation of host cells into specialized feeding cells.

The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection.

Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining.

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means.

Three BUB1 and BUBR1/MAD3-related spindle assembly checkpoint proteins are required for accurate mitosis in Arabidopsis.

The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment.