Whole-Genome Sequencing and Analysis of Tumour-Forming Radish (Raphanus sativus L.) Line.

Spontaneous tumour formation in higher plants can occur in the absence of pathogen invasion, depending on the plant genotype. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this paper, using Oxford Nanopore and Illumina technologies, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We identified a large number of single nucleotide variants (amino acid substitutions, insertions or deletions, SNVs) that are likely to be associated with the spontaneous tumour formation. Among the genes involved in the trait, we have identified those that regulate the cell cycle, meristem activity, gene expression, and metabolism and signalling of phytohormones. After identifying the SNVs, we performed Sanger sequencing of amplicons corresponding to SNV-containing regions to validate our results. We then checked for the presence of SNVs in other tumour lines of the radish genetic collection and found the ERF118 gene, which had the SNVs in the majority of tumour lines. Furthermore, we performed the identification of the CLAVATA3/ESR (CLE) and WUSCHEL (WOX) genes and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

Integrated Transcriptome and Metabolome Dynamic Analysis of Galls Induced by the Gall Mite Aceria pallida on Lycium barbarum Reveals the Molecular Mechanism Underlying Gall Formation and Development.

Galls have become the best model for exploring plant-gall inducer relationships, with most studies focusing on gall-inducing insects but few on gall mites. The gall mite Aceria pallida is a major pest of wolfberry, usually inducing galls on its leaves. For a better understanding of gall mite growth and development, the dynamics of the morphological and molecular characteristics and phytohormones of galls induced by A. pallida were studied by histological observation, transcriptomics and metabolomics. The galls developed from cell elongation of the epidermis and cell hyperplasia of mesophylls. The galls grew quickly, within 9 days, and the mite population increased rapidly within 18 days. The genes involved in chlorophyll biosynthesis, photosynthesis and phytohormone synthesis were significantly downregulated in galled tissues, but the genes associated with mitochondrial energy metabolism, transmembrane transport, carbohydrates and amino acid synthesis were distinctly upregulated. The levels of carbohydrates, amino acids and their derivatives, and indole-3-acetic acid (IAA) and cytokinins (CKs), were markedly enhanced in galled tissues. Interestingly, much higher contents of IAA and CKs were detected in gall mites than in plant tissues. These results suggest that galls act as nutrient sinks and favor increased accumulation of nutrients for mites, and that gall mites may contribute IAA and CKs during gall formation.

Agrobacterium-mediated gene transfer: recent advancements and layered immunity in plants.

MAIN CONCLUSION: Plant responds to Agrobacterium via three-layered immunity that determines its susceptibility or resistance to Agrobacterium infection. Agrobacterium tumefaciens is a soil-borne Gram-negative bacterium that causes crown gall disease in plants. The remarkable feat of interkingdom gene transfer has been extensively utilised in plant biotechnology to transform plant as well as non-host systems. In the past two decades, the molecular mode of the pathogenesis of A. tumefaciens has been extensively studied. Agrobacterium has also been utilised as a premier model to understand the defence response of plants during plant-Agrobacterium interaction. Nonetheless, the threat of Agrobacterium-mediated crown gall disease persists and is associated with a huge loss of plant vigour in agriculture. Understanding the molecular dialogues between these two interkingdom species might provide a cure for crown gall disease. Plants respond to A. tumefaciens by mounting a three-layered immune response, which is manipulated by Agrobacterium via its virulence effector proteins. Comparative studies on plant defence proteins versus the counter-defence of Agrobacterium have shed light on plant susceptibility and tolerance. It is possible to manipulate a plant's immune system to overcome the crown gall disease and increase its competence via A. tumefaciens-mediated transformation. This review summarises the recent advances in the molecular mode of Agrobacterium pathogenesis as well as the three-layered immune response of plants against Agrobacterium infection.

Agrobacterium tumefaciens fitness genes involved in the colonization of plant tumors and roots.

Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots. The identified fitness genes represent 3-8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope-associated functions. Competition assays between 12 knockout mutants and wild-type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation. This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen.

A tale of two tissues: Probing gene expression in a complex insect-induced gall.

Plant galls are novel and sometimes dramatic plant organs whose development is initiated and controlled by parasitic microbes, nematodes, insects and mites. For arthropods, galls provide relative safety from enemies and abiotic stresses while providing nutrition. Galls are formed entirely by the plant, whose transcriptional pathways are modified and coopted to produce a structure specific to the galler species; they comprise a classic example of Dawkins' "extended phenotype". Arthropod-elicited galls are unique in that they are often anatomically complex (Figure 1a), with multiple differentiated tissue types (Figure 1b). A growing number of investigators have studied changes in hostplant gene expression to understand arthropod gall development. In this issue of Molecular Ecology, Martinson et al. (2021) report using RNA sequencing to explore tissue-specific gene expression associated with anatomical and functional gall complexity, demonstrating for the first time that gall tissues are as different transcriptionally as they are anatomically.

Tissue-specific gene expression shows a cynipid wasp repurposes oak host gene networks to create a complex and novel parasite-specific organ.

Every organism on Earth depends on interactions with other organisms to survive. In each of these interactions, an organism must utilize the limited toolbox of genes and proteins it possesses to successfully manipulate or cooperate with another species, but it can also co-opt the genome machinery of its partner to expand its available tools. Insect-induced plant galls are an extreme example of this, wherein an insect hijacks the plant's genome to direct the initiation and development of galls consisting of plant tissue. However, previous transcriptomic studies have not evaluated individual tissues within a gall to determine the full extent to which a galling insect manipulates its host plant. Here we demonstrate that the cynipid wasp Dryocosmus quercuspalustris creates a complex parasite-specific organ from red oak tissue via massive changes in host gene expression. Our results show that the gall wasp is not merely modifying oak leaf tissue but creating extensive changes in gene expression between galled and ungalled tissue (differential expression in 28% of genes) and distinct gall tissue types (20% of genes). The outer gall tissue shows increases in various plant defence systems, which is consistent with its predicted functional role of protecting the wasp larva. The inner larval capsule shows suppression of large parts of the plant innate immune system and evidence for the wasp utilizing the plant's RNA interference mechanisms, which may be a potential mechanism for the wasp's control on gall growth.

Evolutionary classification of tumor- and root-inducing plasmids based on T-DNAs and virulence regions.

Tumor-inducing (Ti) and root-inducing (Ri) plasmids of Agrobacterium that display a large diversity are involved in crown gall and hairy root plant diseases. Their phylogenetic relationships were inferred from an exhaustive set of Ti and Ri plasmids (including 36 new complete Ti plasmids) by focusing on T-DNA and virulence regions. The opine synthase gene content of T-DNAs revealed 13 opine types corresponding to former classifications based on opines detected in diseased plants, while the T-DNA gene content more finely separate opine types in 18 T-DNA organizations. This classification was supported by the phylogeny of T-DNA oncogenes of Ti plasmids. The five gene organizations found in Ti/Ri vir regions was supported by the phylogeny of common vir genes. The vir organization was found to be likely an ancestral plasmid trait separating "classic" Ti plasmids (with one or two T-DNAs) and "Ri and vine-Ti" plasmids. A scenario generally supported by the repABC phylogeny. T-DNAs likely evolved later with the acquisition of opine characteristics as last steps in the Ti/Ri plasmid evolution. This novel evolutionary classification of Ti/Ri plasmids was found to be relevant for accurate crown gall and hairy root epidemiology.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Molecular and Histologic Adaptation of Horned Gall Induced by the Aphid Schlechtendalia chinensis (Pemphigidae).

Chinese galls are the result of hyperplasia in host plants induced by aphids. The metabolism and gene expression of these galls are modified to accommodate the aphids. Here, we highlight the molecular and histologic features of horned galls according to transcriptome and anatomical structures. In primary pathways, genes were found to be unevenly shifted and selectively expressed in the galls and leaves near the galls (LNG). Pathways for amino acid synthesis and degradation were also unevenly shifted, favoring enhanced accumulation of essential amino acids in galls for aphids. Although galls enhanced the biosynthesis of glucose, which is directly available to aphids, glucose content in the gall tissues was lower due to the feeding of aphids. Pathways of gall growth were up-regulated to provide enough space for aphids. In addition, the horned gall has specialized branched schizogenous ducts and expanded xylem in the stalk, which provide a broader feeding surface for aphids and improve the efficiency of transportation and nutrient exchange. Notably, the gene expression in the LNG showed a similar pattern to that of the galls, but on a smaller scale. We suppose the aphids manipulate galls to their advantage, and galls lessen competition by functioning as a medium between the aphids and their host plants.

Mixed Transcriptome Analysis Revealed the Possible Interaction Mechanisms between Zizania latifolia and Ustilago esculenta Inducing Jiaobai Stem-Gall Formation.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta, which may conduct fungus growth and infection at the initial stage of stem-gall formation.

Comparative Transcriptome Analysis of Rutabaga (Brassica napus) Cultivars Indicates Activation of Salicylic Acid and Ethylene-Mediated Defenses in Response to Plasmodiophora brassicae.

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important soilborne disease of Brassica napus L. and other crucifers. To improve understanding of the mechanisms of resistance and pathogenesis in the clubroot pathosystem, the rutabaga (B. napus subsp. rapifera Metzg) cultivars 'Wilhelmsburger' (resistant) and 'Laurentian' (susceptible) were inoculated with P. brassicae pathotype 3A and their transcriptomes were analyzed at 7, 14, and 21 days after inoculation (dai) by RNA sequencing (RNA-seq). Thousands of transcripts with significant changes in expression were identified in each host at each time-point in inoculated vs. non-inoculated plants. Molecular responses at 7 and 14 dai supported clear differences in the clubroot response mechanisms of the two genotypes. Both the resistant and the susceptible cultivars activated receptor-like protein (RLP) genes, resistance (R) genes, and genes involved in salicylic acid (SA) signaling as clubroot defense mechanisms. In addition, genes related to calcium signaling and genes encoding leucine-rich repeat (LRR) receptor kinases, the respiratory burst oxidase homolog (RBOH) protein, and transcription factors such as WRKYs, ethylene responsive factors, and basic leucine zippers (bZIPs), appeared to be upregulated in 'Wilhelmsburger' to restrict P. brassicae development. Some of these genes are essential components of molecular defenses, including ethylene (ET) signaling and the oxidative burst. Our study highlights the importance of activation of genes associated with SA- and ET-mediated responses in the resistant cultivar. A set of candidate genes showing contrasting patterns of expression between the resistant and susceptible cultivars was identified and includes potential targets for further study and validation through approaches such as gene editing.

Molecular Changes Concomitant with Vascular System Development in Mature Galls Induced by Root-Knot Nematodes in the Model Tree Host Populus tremula × P. alba.

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita, a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls.

Secretory RING finger proteins function as effectors in a grapevine galling insect.

BACKGROUND: All eukaryotes share a conserved network of processes regulated by the proteasome and fundamental to growth, development, or perception of the environment, leading to complex but often predictable responses to stress. As a specialized component of the ubiquitin-proteasome system (UPS), the RING finger domain mediates protein-protein interactions and displays considerable versatility in regulating many physiological processes in plants. Many pathogenic organisms co-opt the UPS through RING-type E3 ligases, but little is known about how insects modify these integral networks to generate novel plant phenotypes. RESULTS: Using a combination of transcriptome sequencing and genome annotation of a grapevine galling species, Daktulosphaira vitifoliae, we identified 138 putatively secretory protein RING-type (SPRINGs) E3 ligases that showed structure and evolutionary signatures of genes under rapid evolution. Moreover, the majority of the SPRINGs were more expressed in the feeding stage than the non-feeding egg stage, in contrast to the non-secretory RING genes. Phylogenetic analyses indicated that the SPRINGs formed clusters, likely resulting from species-specific gene duplication and conforming to features of arthropod host-manipulating (effector) genes. To test the hypothesis that these SPRINGs evolved to manipulate cellular processes within the plant host, we examined SPRING interactions with grapevine proteins using the yeast two-hybrid assay. An insect SPRING interacted with two plant proteins, a cellulose synthase, CSLD5, and a ribosomal protein, RPS4B suggesting secretion reprograms host immune signaling, cell division, and stress response in favor of the insect. Plant UPS gene expression during gall development linked numerous processes to novel organogenesis. CONCLUSIONS: Taken together, D. vitifoliae SPRINGs represent a novel gene expansion that evolved to interact with Vitis hosts. Thus, a pattern is emerging for gall forming insects to manipulate plant development through UPS targeting.

Improved genetic resolution for linkage mapping of resistance to potato wart in monoparental dihaploids with potential diagnostic value in tetraploid potato varieties.

KEY MESSAGE: We achieved improved mapping resolution of the major wart resistance locus Xla-TNL containing also Sen1 in a dihaploid population using SNP data and developed additional markers with diagnostic value in tetraploid varieties. We analyzed a segregating monoparental dihaploid potato population comprising 215 genotypes derived from a tetraploid variety that is highly resistant to Synchytrium endobioticum pathotypes 18 and 6. The clear bimodal segregation for both pathotypes indicated that a major dominant resistance factor in a simplex allele configuration was present in the tetraploid donor genotype. Compared to that in previous analyses of the same tetraploid donor in conventional crosses with susceptible tetraploid genotypes, a segregation pattern with a reduced genetic complexity of resistance in dihaploids was observed here. Using the 12.8 k SolCAP SNP array, we mapped a resistance locus to the Xla-TNL region containing also Sen1 on potato chromosome 11. The improved mapping resolution provided by the monoparental dihaploids allowed for the localization of the genes responsible for the resistance to both pathotypes in an interval spanning less than 800 kbp on the reference genome. Furthermore, we identified eight molecular markers segregating without recombination to pathotype 18 and pathotype 6 resistance. Also, two developed markers display improved diagnostic properties in an independent panel of tetraploid varieties. Overall, our data provide the highest resolution mapping of wart resistance genes at the Xla-TNL locus thus far.

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells' area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

Exploiting cell cycle inhibitor genes of the KRP family to control root-knot nematode induced feeding sites in plants.

Cell cycle control in galls provoked by root-knot nematodes involves the activity of inhibitor genes like the Arabidopsis ICK/KRP members. Ectopic KRP1, KRP2 and KRP4 expression resulted in decreased gall size by inhibiting mitotic activity, whereas KRP6 induces mitosis in galls. Herein, we investigate the role of KRP3, KRP5 and KRP7 during gall development and compared their role with previously studied members of this class of cell cycle inhibitors. Overexpression of KRP3 and KRP7 culminated in undersized giant cells, with KRP3OE galls presenting peculiar elongated giant cells. Nuclei in KRP3OE and KRP5OE lines presented a convoluted and apparently connected phenotype. This appearance may be associated with the punctuated protein nuclear localization driven by specific common motifs. As well, ectopic expression of KRP3OE and KRP5OE affected nematode development and offspring. Decreased mitotic activity in galls of KRP3OE and KRP7OE lines led to a reduced gall size which presented distinct shapes - from more elongated like in the KRP3OE line to small rounded like in the KRP7OE line. Results presented strongly support the idea that induced expression of cell cycle inhibitors such as KRP3 and KRP7 in galls can be envisaged as a conceivable strategy for nematode feeding site control in crop species attacked by phytopathogenic nematodes.

Transcriptomic characterization of gall tissue of Japanese elm tree (Ulmus davidiana var. japonica) induced by the aphid Tetraneura nigriabdominalis.

Insect galls are abnormal plant tissues induced by parasitic insect(s) for use as their habitat. In previous work, we suggested that gall tissues induced by the aphid Tetraneura nigriabdominalis on Japanese elm trees are less responsive than leaf tissues to jasmonic acid (JA), which is involved in the production of volatile organic compounds as a typical defensive reaction of plants against attack by insect pests. A comprehensive analysis of gene expression by RNA sequencing indicated that the number of JA responsive genes was markedly lower in gall tissues than in leaf tissues. This suggests that gall tissues are mostly defective in JA signaling, although JA signaling is not entirely compromised in gall tissue. Gene ontology analysis sheds light on some stress-related unigenes with higher expression levels in gall tissues, suggesting that host plants sense aphids as a biotic stress but are defective in the JA-mediated defense response in gall tissues.

Expression and evolutionary analyses of three acetylcholinesterase genes (Mi-ace-1, Mi-ace-2, Mi-ace-3) in the root-knot nematode Meloidogyne incognita.

The full cDNA of Mi-ace-3 encoding an acetylcholinesterase (AChE) in Meloidogyne incognita was cloned and characterized. Mi-ace-3 had an open reading frame of 1875 bp encoding 624 amino acid residues. Key residues essential to AChE structure and function were conserved. The deduced Mi-ACE-3 protein sequence had 72% amino acid similarity with that of Ditylenchus destructor Dd-AChE-3. Phylogenetic analyses using 41 AChEs from 24 species showed that Mi-ACE-3 formed a cluster with 4 other nematode AChEs. Our results revealed that the Mi-ace-3 cloned in this study, which is orthologous to Caenorhabditis elegans AChE, belongs to the nematode ACE-3/4 subgroup. There was a significant reduction in the number of galls in transgenic tobacco roots when Mi-ace-1, Mi-ace-2, and Mi-ace-3 were knocked down simultaneously, whereas little or no effect were observed when only one or two of these genes were knocked down. This is an indication that the functions of these three genes are redundant.

The plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.

In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified plants and bacteria, we evaluated the impact of plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type tumors relative to uninfected plant tissues. Moreover, comparisons of tumors induced on Col-0 and her1 plants showed that the increase in the plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in plant tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights plant GABA content as an important trait in the struggle against pathogenic bacteria.

Differentially expressed small RNAs in Arabidopsis galls formed by Meloidogyne javanica: a functional role for miR390 and its TAS3-derived tasiRNAs.

Root-knot nematodes (RKNs) induce inside the vascular cylinder the giant cells (GCs) embedded in the galls. The distinctive gene repression in early-developing GCs could be facilitated by small RNAs (sRNA) such as miRNAs, and/or epigenetic mechanisms mediated by 24nt-sRNAs, rasiRNAs and 21-22nt-sRNAs. Therefore, the sRNA-population together with the role of the miR390/TAS3/ARFs module were studied during early gall/GC formation. Three sRNA libraries from 3-d-post-inoculation (dpi) galls induced by Meloidogyne javanica in Arabidopsis and three from uninfected root segments were sequenced following Illumina-Solexa technology. pMIR390a::GUS and pTAS3::GUS lines were assayed for nematode-dependent promoter activation. A sensor line indicative of TAS3-derived tasiRNAs binding to the ARF3 sequence (pARF3:ARF3-GUS) together with a tasiRNA-resistant ARF3 line (pARF3:ARF3m-GUS) were used for functional analysis. The sRNA population showed significant differences between galls and controls, with high validation rate and correspondence with their target expression: 21-nt sRNAs corresponding mainly to miRNAs were downregulated, whilst 24-nt-sRNAs from the rasiRNA family were mostly upregulated in galls. The promoters of MIR390a and TAS3, active in galls, and the pARF3:ARF3-GUS line, indicated a role of TAS3-derived-tasiRNAs in galls. The regulatory module miR390/TAS3 is necessary for proper gall formation possibly through auxin-responsive factors, and the abundance of 24-nt sRNAs (mostly rasiRNAs) constitutes a gall hallmark.