OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast.

With the aim of identifying novel regulators of host and nonhost resistance to fungi in rice, we carried out a systematic mutant screen of mutagenized lines. Two mutant wrky22 knockout lines revealed clear-cut enhanced susceptibility to both virulent and avirulent Magnaporthe oryzae strains and altered cellular responses to nonhost Magnaporthe grisea and Blumeria graminis fungi. In addition, the analysis of the pathogen responses of 24 overexpressor OsWRKY22 lines revealed enhanced resistance phenotypes on infection with virulent M. oryzae strain, confirming that OsWRKY22 is involved in rice resistance to blast. Bioinformatic analyses determined that the OsWRKY22 gene belongs to a well-defined cluster of monocot-specific WRKYs. The co-regulatory analysis revealed no significant co-regulation of OsWRKY22 with a representative panel of OsWRKYs, supporting its unique role in a series of transcriptional responses. In contrast, inquiring a subset of biotic stress-related Affymetrix data, a large number of resistance and defence-related genes were found to be putatively co-expressed with OsWRKY22. Taken together, all gathered experimental evidence places the monocot-specific OsWRKY22 gene at the convergence point of signal transduction circuits in response to both host and nonhost fungi encountering rice plants.

Building a mutant resource for the study of disease resistance in rice reveals the pivotal role of several genes involved in defence.

In Arabidopsis, gene expression studies and analysis of knock-out (KO) mutants have been instrumental in building an integrated view of disease resistance pathways. Such an integrated view is missing in rice where shared tools, including genes and mutants, must be assembled. This work provides a tool kit consisting of informative genes for the molecular characterization of the interaction of rice with the major fungal pathogen Magnaporthe oryzae. It also provides for a set of eight KO mutants, all in the same genotypic background, in genes involved in key steps of the rice disease resistance pathway. This study demonstrates the involvement of three genes, OsWRKY28, rTGA2.1 and NH1, in the establishment of full basal resistance to rice blast. The transcription factor OsWRKY28 acts as a negative regulator of basal resistance, like the orthologous barley gene. Finally, the up-regulation of the negative regulator OsWRKY28 and the down-regulation of PR gene expression early during M. oryzae infection suggest that the fungus possesses infection mechanisms that enable it to block host defences.

Characterization of WRKY co-regulatory networks in rice and Arabidopsis.

BACKGROUND: The WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data. RESULTS: The presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes. CONCLUSION: In this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.

Characterization of the model system rice--Magnaporthe for the study of nonhost resistance in cereals.

The best characterized form of resistance is gene-for-gene resistance. Less well characterized is nonhost resistance in which an entire plant species is resistant to an entire pathogen species. Here, different rice genotypes were inoculated with host and nonhost strains of Magnaporthe isolated from rice, wheat and crabgrass. The different types of interactions were characterized at a cytological level using a 3,3'-diaminobenzidine (DAB) stain to investigate the occurrence of reactive oxygen intermediates or by observing the occurrence of cellular autofluorescence. Gene expression of a set of selected PR-genes was analysed using quantitative real-time polymerase chain reaction. Inoculation with the isolate from crabgrass resulted in a lack of penetration. The wheat isolate induced a hypersensitive response with varying degrees of pathogen growth inside the invaded cell according to the rice genotype. Expression analysis of our PR-gene set revealed clear differences between the different types of interactions in both kinetic and magnitude of gene induction. Our integrated study opens the way to the dissection of molecular components leading to nonhost reactions to Magnaporthe grisea in rice and points to novel sources of durable resistance to fungal plant pathogens in other cereal crops.

Effect of NaCl and mannitol iso-osmotic stresses on proline and free polyamine levels in embryogenic Fraxinus angustifolia callus.

With the aim to differentiate the ionic and osmotic components of salt stress, short and long-term changes in free polyamines and proline induced by iso-osmotic concentrations of NaCl (0.1 mol/L and 0.2 mol/L) and mannitol (0.2 mol/L and 0.4 mol/L) were determined in Fraxinus angustifolia callus. The peculiarities of the short-term responses were: i) a very early (30 min) and temporary increase in Putrescine (Pu) and Spermine (Spm) as a consequence of salt treatment, and ii) a continuous accumulation of Spermidine (Spd) and Spm in response to mannitol. The changes of Proline (Pro) were quite limited both in the short and in the long term, and generally occurred later than Polyamine (PAs) changes took place, suggesting a regulatory mechanism of PAs metabolism on Pro biosynthesis. In the long-term, no drastic accumulations of Pro or PAs in response to NaCl and mannitol were observed, suggesting that their physiological role is unlikely to be that of osmo-compatible solutes in this plant system. The salt induced a higher callus growth inhibition effect than did mannitol and this inhibition was associated with the reduction of endogenous levels of PAs, especially Pu. However, while a diverging time course was observed under lethal salt concentration (0.2 mol/L NaCl), a high parallelism in the endogenous changes of Pro and Pu was observed under all non-lethal conditions (control--0.2 and 0.4 mol/L mannitol--0.1 mol/L NaCl). Therefore the synchronous changes of Pro and Pu can be considered as a physiological trait associated with cell survival. These results indicate a strong metabolic co-ordination between PAs and Pro pathways and suggest that the metabolic fluxes through these pathways start competing only when the stress level is high enough to be lethal for cells.

Potato virus X-induced gene silencing in leaves and tubers of potato.

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.

Regulated expression of a novel TCP domain transcription factor indicates an involvement in the control of meristem activation processes in Solanum tuberosum.

In this study, the aim was to determine whether TCP transcription factors are implicated in meristem activation in potato (Solanum tuberosum). By searching a database of potato EST sequences, with a sequence characteristically conserved in TCP domains, a potato tcp gene was identified. A BAC clone containing the tcp sequence was isolated and the genomic sequence was determined. Using a CAPS marker assay, the potato tcp gene (sttcp1) was mapped to chromosome 8. In dormant buds, relatively high levels of sttcp1-specific transcript were detected by in situ hybridization. By contrast, in sprouting buds, no expression of the sttcp1 could be detected. Furthermore, an inverse relationship between axillary bud size and the steady-state level of the sstcp1 transcript was demonstrated. In non-growing buds exhibiting correlative inhibition, sttcpI-specific transcript levels were also relatively high, but rapidly decreased when apical dominance was removed by excision of the apical bud.

Changes in gene expression during meristem activation processes in Solanum tuberosum with a focus on the regulation of an auxin response factor gene.

A suppression subtractive hybridization approach (SSH) was used to generate a cDNA library enriched in clones representing genes that are up-regulated in the potato tuber apical bud on dormancy release. The sequences of cDNAs representing 385 different genes were determined. This study focuses on the characterization of one of these cDNAs. On the basis of sequence similarity, the cDNA was identified as encoding a member of the auxin response factor family (ARF6). The expression pattern of potato ARF6 was determined by in situ hybridization. In apical tuber buds in the early stages of sprouting, relatively high levels of ARF6-specific transcripts were detected, especially in the peripheral zones of the tunica and corpus of the apical meristems. Expression was also detected in procambial and early vascular tissues, both subtending the meristem and in adjacent leaf primordia. By contrast, in dormant buds no expression of ARF6 could be detected. The expression pattern was also determined during the tuberization process; steady-state expression levels decreased c. 10-fold in the apical region as tuberization proceeded. In non-growing buds, exhibiting correlative inhibition, ARF6-specific transcript levels were relatively low, but rapidly increased when apical dominance was removed by excision of the apical bud. The effects of gibberellin and auxin on axillary bud growth and ARF6 expression are described.

Cuttings of the non-rooting rac tobacco mutant overaccumulate phenolic compounds.

The auxin and phenolic contents, as well as phenylalanine ammonia-lyase (PAL) activity, were determined in in vitro cultured shoots of the recalcitrant-to-root rac mutant of tobacco, and compared with wild-type shoots. The mutant and wild-type shoots showed similar auxin changes during the culture cycle, but with higher contents for the mutant. A transient peak of auxin (corresponding to the achievement of the rooting inductive phase) occurred at day 14 in both types of shoots, but earlier in the basal parts of the wild-type stems. The rac shoots contained more phenolics, corresponding with an increased PAL activity. The most abundant phenolic compound found in the two types of tobacco was chlorogenic acid, which was more abundant in the rac shoots. Rutin was also detected at a higher concentration in the mutant shoots. Basal parts of wild-type shoots treated with 10-3 M chlorogenic acid reacted by accumulating auxins and, unlike untreated controls, did not form adventitious roots. The relationships between these biochemical analyses in relation to the growth limitation of the rac mutant, and the inhibition of its root development, are discussed.