Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis.

The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Sensitivity of Yeast Mutants Deficient in Mitochondrial or Vacuolar ABC Transporters to Pathogenesis-Related Protein TcPR-10 of Theobroma cacao.

Pathogenesis-related proteins (PRs) are induced in plants after infection by pathogens and/or abiotic stress. Among these proteins, the family 10 (PR-10) influences the biosynthesis of secondary metabolites and shows antimicrobial ribonuclease activity. TcPR-10p (Pathogenesis-related Protein 10 of Theobroma cacao) was isolated from resistant and susceptible Moniliophthora perniciosa cacao cultivars. Cell survival with Saccharomyces cerevisiae mutant lines deficient in ATP-binding cassette (ABC) transporter proteins indicated the influence on resistance to TcPR-10p. Proteins of the ABC transport type are considered important in the process of resistance to antimicrobials and toxins. Thus, the objective of this work was to observe the sensitivity of ABC transporter yeast mutants in the presence of the TcPR-10p. Chronic exposure of S. cerevisiae mitochondrial (BYatm1Δ and BYmdl1Δ) and vacuole (BYnft1Δ, BYvmr1Δ, BYybt1Δ, BYycf1Δ and BYbpt1Δ) ABC transporter mutants to TcPR-10p (3 μg/mL, 0, 6, 12 and 24 h) was performed. Two TcPR-10p sensitive strains (BYmdl1Δ and BYnft1Δ) were submitted to a fluorescence test with the fluorogenic dihydroethidium (DHE), to visualize the presence of oxidative stress in the cells. Oxidative stress-increased sensitivity was confirmed by flow cytometry indicating induced cell death either via apoptosis or necrosis. This yeast data combined with previous data of literature (of M. perniciosa sensitivity to TcPR-10p) show that increased sensitivity to TcPR-10p in these mutants could be due to the TcPR10p-generated higher levels of intracellular reactive oxygen species (ROS), leading to increased cell death either via necrosis or apoptosis.

Cloning, expression and purification of kinase domains of cacao PR-1 receptor-like kinases.

The PR-1 proteins (pathogenesis-related protein 1) are involved in plant defense mechanisms against various pathogens. The genome of cacao (Theobroma cacao) encodes 14 PR-1 proteins, named TcPR-1a to TcPR-1n. Two of them, TcPR-1f and TcPR-1g, have a C-terminal expansion with high similarity to protein kinase domains, suggesting a receptor-like kinase (RLK) protein architecture. Moreover, TcPR-1g is highly expressed during cacao response to Witches' Broom Disease, caused by the fungus Moniliopthora perniciosa. Here we describe a structural genomics approach to clone, express and purify the kinase domains of TcPR-1f and TcPR-1g. Escherichia coli BL21(DE3)-R3 cells were used for protein expression and co-expression of Lambda Protein Phosphatase was critical for successfully obtaining soluble recombinant protein. We expect that the ability to express and purify the kinase domains of TcPR-1f and TcPR-1g will further our understanding of the role these proteins play during cacao defense response.

Cloning and Expression Analysis of HbPR-1b and HbPR-3 in Hevea brasiliensis During Inoculation with Rigidoporus microporus.

BACKGROUND AND OBJECTIVE: Pathogenesis-related (PR) proteins are dramatically accumulated after pathogen infection. Innate defense response through increasing PR-proteins is important for rubber rootstock selection that is tolerant to the white root disease caused by Rigidoporus microporus. This study was aimed to investigate the expression levels of PR-1 and PR-3 genes in tolerant (PB5/51) and susceptible (BPM24 and RRIM600) rubber clones after R. microporus infection. MATERIALS AND METHODS: The mRNA of HbPR-1b and HbPR-3 was isolated and characterized from rubber leaves. Gene expression levels of HbPR-1b and HbPR-3 were compared among three rubber clones (PB5/51, BPM24 and RRIM600) after R. microporus infection at 0, 12, 24, 48, 72 and 96 h using quantitative real-time PCR. The relative transcript abundances between inoculated and control plants were compared using the means of gene expression between time points and by Tukey's HSD test. A probability value (p<0.05) was used to determine the significance of difference between time points. RESULTS: The open reading frame of HbPR-1b is 492 bp with deduced 163 amino acid residues and the phylogenetic analysis showed it shared significant evolutionary history and clustering into group I of PR-protein. Moreover, the partial HbPR-3 was isolated with 390 bp. Gene expression levels of HbPR-1b and HbPR-3 showed marked differences in both transcripts depending on the rubber clones. Two genes demonstrated up-regulation of both tolerance and susceptibility in response to attack by R. microporus. The highest expression levels were found in seedlings of PB5/51 after inoculation. In RRIM600, low expression levels of HbPR-1b and HbPR-3 were initially observed but gradually increased at 24 h post inoculation. The transcription profile of HbPR-1b was stable expression in BPM24. CONCLUSION: The results demonstrated that the level ofHbPR-1b and HbPR-3 transcription can distinguish between tolerant and susceptible clones. The candidate defense genes to the white root disease were observed in PB5/51 seedlings, particularly HbPR-1b.

Moss Pathogenesis-Related-10 Protein Enhances Resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana.

Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.