Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

The root-knot nematode calreticulin Mi-CRT is a key effector in plant defense suppression.

Root-knot nematodes (RKN) are obligate biotrophic parasites that settle close to the vascular tissues in roots, where they induce the differentiation of specialized feeding cells and maintain a compatible interaction for 3 to 8 weeks. Transcriptome analyses of the plant response to parasitic infection have shown that plant defenses are strictly controlled during the interaction. This suggests that, similar to other pathogens, RKN secrete effectors that suppress host defenses. We show here that Mi-CRT, a calreticulin (CRT) secreted by the nematode into the apoplasm of infected tissues, plays an important role in infection success, because Mi-CRT knockdown by RNA interference affected the ability of the nematodes to infect plants. Stably transformed Arabidopsis thaliana plants producing the secreted form of Mi-CRT were more susceptible to nematode infection than wild-type plants. They were also more susceptible to infection with another root pathogen, the oomycete Phytophthora parasitica. Mi-CRT overexpression in A. thaliana suppressed the induction of defense marker genes and callose deposition after treatment with the pathogen-associated molecular pattern elf18. Our results show that Mi-CRT secreted in the apoplasm by the nematode has a role in the suppression of plant basal defenses during the interaction.

Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode.

Root-knot nematodes (RKNs) are sedentary biotrophic parasites that induce the differentiation of root cells into feeding cells that provide the nematodes with the nutrients necessary for their development. The development of new control methods against RKNs relies greatly on the functional analysis of genes that are crucial for the development of the pathogen or the success of parasitism. In the absence of genetic transformation, RNA interference (RNAi) allows for phenotype analysis of nematode development and nematode establishment in its host after sequence-specific knock-down of the targeted genes. Strategies used to induce RNAi in RKNs are so far restricted to small-scale analyses. In the search for a new RNAi strategy amenable to large-scale screenings the possibility of using RNA viruses to produce the RNAi triggers in plants was tested. Tobacco rattle virus (TRV) was tested as a means to introduce double-stranded RNA (dsRNA) triggers into the feeding cells and to mediate RKN gene silencing. It was demonstrated that virus-inoculated plants can produce dsRNA and siRNA silencing triggers for delivery to the feeding nematodes. Interestingly, the knock-down of the targeted genes was observed in the progeny of the feeding nematodes, suggesting that continuous ingestion of dsRNA triggers could be used for the functional analysis of genes involved in early development. However, the heterogeneity in RNAi efficiency between TRV-inoculated plants appears as a limitation to the use of TRV-mediated silencing for the high-throughput functional analysis of the targeted nematode genes.

RNAi and functional genomics in plant parasitic nematodes.

Plant nematology is currently undergoing a revolution with the availability of the first genome sequences as well as comprehensive expressed sequence tag (EST) libraries from a range of nematode species. Several strategies are being used to exploit this wealth of information. Comparative genomics is being used to explore the acquisition of novel genes associated with parasitic lifestyles. Functional analyses of nematode genes are moving toward larger scale studies including global transcriptome profiling. RNA interference (RNAi) has been shown to reduce expression of a range of plant parasitic nematode genes and is a powerful tool for functional analysis of nematode genes. RNAi-mediated suppression of genes essential for nematode development, survival, or parasitism is revealing new targets for nematode control. Plant nematology in the genomics era is now facing the challenge to develop RNAi screens adequate for high-throughput functional analyses.

Transcriptome analysis of root-knot nematode functions induced in the early stages of parasitism.

Root-knot nematodes of the genus Meloidogyne are obligate biotrophic parasites able to infest > 2000 plant species. The nematode effectors responsible for disease development are involved in the adaptation of the parasite to its host environment and host response modulation. Here, the differences between the transcriptomes of preparasitic exophytic second-stage juveniles (J2) and parasitic endophytic third-stage juveniles (J3) of Meloidogyne incognita were investigated. Genes up-regulated at the endophytic stage were isolated by suppression subtractive hybridization and validated by dot blots and real-time quantitative polymerase chain reaction (PCR). Up-regulation was demonstrated for genes involved in detoxification and protein degradation, for a gene encoding a putative secreted protein and for genes of unknown function. Transcripts of the glutathione S-transferase gene Mi-gsts-1 were 27 times more abundant in J3 than in J2. The observed Mi-gsts-1 expression in the oesophageal secretory glands and the results of functional analyses based on RNA interference suggest that glutathione S-transferases are secreted during parasitism and are required for completion of the nematode life cycle in its host. Secreted glutathione S-transferases may protect the parasite against reactive oxygen species or modulate the plant responses triggered by pathogen attack.

Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins.

Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the root-knot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4 h induced double-stranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20 h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

Comparative analysis of two 14-3-3 homologues and their expression pattern in the root-knot nematode Meloidogyne incognita.

14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells.

Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita.

Amplified fragment length polymorphism fingerprinting of three pairs of Meloidogyne incognita near-isogenic lines (NILs) was used to identify markers differential between nematode genotypes avirulent or virulent against the tomato Mi resistance gene. One of these sequences, present only in the avirulent lines, was used as a probe to screen a cDNA library from second-stage juveniles (J2s) and allowed cloning of a cDNA encoding a secretory protein. The putative full-length cDNA, named map-1, encoded a 458 amino acid (aa) protein containing a predictive N-terminal secretion signal peptide. The MAP-1 sequence did not show any significant similarity to proteins deposited in databases. The internal part of the protein, however, was characterized by highly conserved repetitive motives of 58 or 13 aa. Reverse transcription polymerase chain reaction (RT-PCR) experiments confirmed that map-1 expression was different between avirulent and virulent NILs. In PCR reactions, map-1-related sequences were amplified only in nematode populations belonging to the three species against which the Mi gene confers resistance: M. arenaria, M. incognita, and M. javanica. Polyclonal antibodies raised against a synthetic peptide deduced from the MAP-1 sequence strongly labeled J2 amphidial secretions in immunofluorescence microscopy assays, suggesting that MAP-1 may be involved in the early steps of recognition between (resistant) plants and (avirulent) nematodes.

Biochemical characterization of MI-ENG1, a family 5 endoglucanase secreted by the root-knot nematode Meloidogyne incognita.

A beta-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant-nematode interaction.

Isolation of a cDNA encoding a beta-1,4-endoglucanase in the root-knot nematode Meloidogyne incognita and expression analysis during plant parasitism.

A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.

Molecular cloning of an acetylcholinesterase gene from the plant parasitic nematodes, Meloidogyne incognita and Meloidogyne javanica.

A gene encoding a protein with strong homology with Caenorhabditis elegans and C. briggsae acetylcholinesterase ACE-1 was cloned from Meloidogyne incognita and M. javanica pre-parasitic juveniles. Both cDNAs have an ORF of 1968 bp for a deduced translation product of 656 amino acid residues. The key residues essential to acetylcholinesterase (AChE) structure and function are conserved in both sequences. M. incognita and M. javanica AChE share a homology of 98.8% at the amino acid level and 97% at the nucleotide level. Phylogenetic analysis showed that Meloidogyne and Caenorhabditis AChE form a cluster among AChE of triploblastic organisms. This Meloidogyne AChE is expressed in eggs, pre-parasitic juveniles and males and AChE activity was detected in situ in amphids of pre-parasitic juveniles. The opportunity of using AChE as a target in new strategies of nematode control is discussed.

Improving scFv antibody expression levels in the plant cytosol.

Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.

Expression and functional characterization of a single chain Fv antibody directed against secretions involved in plant nematode infection process.

Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops. Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process. From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv). The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody. The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography. The produced scFv is fully functional since it shows the same specificity towards a crude extract of M. incognita infective larvae as the corresponding parental IgM. Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal. Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants.