Sílvia Tavares

Vitis vinifera secondary metabolism as affected by sulfate depletion: diagnosis through phenylpropanoid pathway genes and metabolites

Grapevine (Vitis vinifera L.) is rich in phenylpropanoid compounds, namely flavonoids and stilbenes which, present in most tissues, are described as antioxidants and known to accumulate in response to biotic and abiotic stress. Grapevine is then a choice model for studying the interplay between the phenylpropanoid pathway and nutrient deficiency. Here we report the response to sulfur deficiency (-S) of flavonoids and stilbenes biosynthetic pathways in chlorophyll tissues (plantlets) and cell culture. Anthocyanins and trans-resveratrol accumulated in plantlets and trans-resveratrol glucoside in cell cultures in response to sulfur deficiency, while a significant decrease in chlorophyll was observed in -S plantlets. The up-regulation of chalcone synthase gene and the downstream flavonoid biosynthesis genes dihydroflavonol reductase and anthocyanidin synthase matched the accumulation of anthocyanins in -S V. vinifera plantlets. The mRNA level of stilbene synthase gene(s) was correlated tightly with the increase in trans-resveratrol and trans-resveratrol glucoside levels, respectively in -S plantlets and cell cultures. As a whole, the present study unveil that V. vinifera under sulfur deficiency allocates resources to the phenylpropanoid pathway, probably consecutive to inhibition of protein synthesis, which can be advantageous to resist against oxidative stress symptoms evoked by -S conditions.

Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix with an emphasis on early stages of infection

Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and compared to previously published in planta haustoria-rich (H) data. A total of 9234 transcripts were identified and annotated. Ca. 50% of these transcripts showed no significant homology to international databases. Only 784 sequences were shared by the three conditions, and 75% were exclusive of either gU (2146), Ap (1479) or H (3270). Relative transcript abundance and RT-qPCR analyses for a selection of genes indicated a particularly active metabolism, translational activity and production of new structures in the appressoria and intense signaling, transport, secretory activity and cellular multiplication in the germinating urediniospores, suggesting the onset of a plant-fungus dialogue as early as at the germ tube stage. Gene expression related to the production of carbohydrate-active enzymes and accumulation of glycerol in germinating urediniospores and appressoria suggests that combined lytic and physical mechanisms are involved in appressoria-mediated penetration. Besides contributing to the characterization of molecular processes leading to appressoria-mediated infection by rust fungi, these results point toward the identification of new H. vastatrix candidate virulence factors, with 516 genes predicted to encode secreted proteins.

Direct detection of Taphrina deformans on peach trees using molecular methods

The ascomycetous fungus Taphrina deformans is the agent of peach leaf curl, a worldwide disease of peach potentially devastating to both crop yields and tree longevity. Conspicuous leaf curl symptoms result from the invasion of host tissue by the strictly parasitic mycelial phase of the T. deformans dimorphic life-cycle. Successful isolation of the fungus in pure culture is cumbersome and limited to late spring/early summer (time of ascospore discharge from infected leaves) and only rarely has the asymptomatic yeast phase been isolated from buds. Molecular methods, namely those based on the hybridisation of nucleic acids, are advantageous for diagnostic purposes since they do not require isolation of the fungus on culture media. Direct amplification using the polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) were tested for diagnosis of peach leaf curl disease in order to provide a fast and reliable method for disease risk assessment. Specific primers and probes were designed based on available ribosomal DNA sequence data. Positive and specific diagnoses of peach leaf curl were achieved with primer TDITS1, using PCR-detection, and probe TDE634, using FISH, both on infected leaves and in washings of asymptomatic peach buds.

Identification and expression of cytokinin signaling and meristem identity genes in sulfur deficient grapevine (Vitis vinifera L.)

In plants, cytokinin (CK) perception and signaling pathway is composed by a histidine kinase receptor (HK) and a response regulator (RR), the signal being mediated by a histidine phosphotransfer (HPt), as described in Arabidopsis, maize and rice. From database searches we identified in grapevine three HKs, three HPs, four A-type RRs and 6 B-type RRs, suggesting a common mechanism for grapevine. The phylogenetic analysis of these Vitis genes showed a variable but high degree of homology with Arabidopsis sequences. When sulfate was withdrawn from the culture medium (-S) of in vitro Vitis shoots, we assessed a significant reduction in shoot branching. To ascertain the crosstalk of S status with CK signaling in grapevine, control and -S grown shoots and control, -S and -CK cell suspensions were used as experimental systems. Real-time PCR was elected to quantify the expression of key genes. The expression of CK receptor genes was down-regulated in -S cells while not affected in -CK cells. In differentiated shoots no response to -S was observed on those genes. A-type VvRRa4 was down-regulated in -S or -CK cells while Vitis B-type RRs did not respond either to CK or S starvation. The results suggest that Vitis CK signaling pathway is affected by -S, although differently according to the model system. Transcription of Vitis apical meristem-identity genes VvWUS, VvCLV and VvSTM and axillary meristem genes VvBRC1, VvBRC2, VvLAS, VvRAX and VvREV was estimated and VvSTM and VvLAS showed to be down-regulated in -S. Then, the expression levels of VvSTM and VvLAS makes them strong candidates to be associated with the branching pattern of Vitis shoots in -S.

Flow cytometry reveals that the rust fungus, Uromyces bidentis (Pucciniales), possesses the largest fungal genome reported—2489 Mbp

Among the Eukaryotes, Fungi have relatively small genomes (average of 44.2 Mbp across 1850 species). The order Pucciniales (Basidiomycota) has the largest average genome size among fungi (305 Mbp), and includes the two largest fungal genomes reported so far (Puccinia chrysanthemi and Gymnosporangium confusum, with 806.5 and 893.2 Mbp, respectively). In this work, flow cytometry was employed to determine the genome size of the Bidens pilosa rust pathogen, Uromyces bidentis. The results obtained revealed that U. bidentis presents a surprisingly large haploid genome size of 2489 Mbp. This value is almost three times larger than the previous largest fungal genome reported and over 50 times larger than the average fungal genome size. Microscopic examination of U. bidentis nuclei also showed that they are not as different in size from the B. pilosa nuclei when compared with the differences between other rusts and their host plants. This result further reinforces the position of the Pucciniales as the fungal group with the largest genomes, prompting studies addressing the role of repetitive elements and polyploidy in the evolution, pathological specialization and diversity of fungal species.

DEREPRESSED SULFATE TRANSPORTERS ARE STRONGLY AND RAPIDLY REPRESSED AFTER SULFATE ADDITION TO SULFUR-DEPLETED VITIS CELLS

A cell system of two Vitis species, Vitis vinifera cv. Touriga Nacional and Vitis rupestris, was chosen to study the response to sulfate deficiency and sulfate resupply through the analysis of sulfate influx and the expression of sulfate transporter transcripts. Cell suspensions were grown under two sulfate conditions: S sufficient (+S; 1.5 mM of MgSO4) and S deficient (−S). Both species were equally affected by the S‐deficient conditions of the medium. After 24 h in −S medium, cells of both species showed a significant increase in sulfate influx rate, which was maintained throughout the culture cycle, reaching a maximum at days 4–5 in −S conditions. The relative expression of sulfate transporters from V. vinifera and V. rupestris, VvST and VrST, analyzed by real‐time PCR, confirmed a strong derepression of the sulfate transporters in −S conditions. The enhanced influx rates and the upregulation of VvST and VrST were rapidly reversed by the addition of \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape

The coffee leaf rust pathogen Hemileia vastatrix: one and a half centuries around the tropics

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Genome size estimates for six rust (Pucciniales) species

\end{document} to the −S medium. Vitis vinifera showed a faster response at the protein level measured by the influx rates, while in V. rupestris, the effect was demonstrated primarily on the relative expression of VrST.