Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and payload proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal beta-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in storage vacuoles.

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane- 1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species. © 2005 Elsevier Ireland Ltd. All rights reserved.

Salicylic acid mediates resistance to the vascular wilt pathogen Fusarium oxysporum in the model host Arabidopsis thaliana

Fusarium oxysporum is a soilborne fungal pathogen that causes major economic losses by inducing necrosis and wilting symptoms in many crop plants. In this study, the interaction between F. oxysporum and the model plant Arabidopsis thaliana has been investigated to better understand the nature of host defences that are effective against the Fusarium wilt pathogen. The expression of salicylate- and jasmonate-responsive defence genes in F. oxysporum-challenged roots of A. thaliana plants as well as in the roots of plants whose leaves were treated with salicylate or jasmonate was analysed. Unexpectedly, genes (e.g. PR1, PDF1.2, and CHIB) encoding proteins with defensive functions or transcription factors (e.g. ERF1, AtERF2, AtERF4 and AtMYC2) known to positively or negatively regulate defences against F. oxysporum were not activated in F. oxysporum-inoculated roots. In contrast, the jasmonate-responsive defence gene PDF1.2 was induced in the leaves of plants whose roots were challenged with F. oxysporum, but the salicylate- responsive PR1 gene was not induced in the leaves of inoculated plants. Exogenous salicylic acid treatment prior to inoculation, however, activated PR1 and BGL2 defence gene expression in leaves and provided increased F. oxysporum resistance as evidenced by reduced foliar necrosis and plant death. Exogenous salicylic acid treatment of the foliar tissue did not activate defence gene expression in the roots of plants. This suggests that salicylate- dependent defences may function in foliar tissue to reduce the development of pathogen-induced wilting and necrosis. Despite the induction of defence gene expression in the leaves by jasmonate, this treatment did not lead to increased resistance to F. oxysporum. Overall, the results presented here suggest that the genetic manipulation of plant defence signalling pathways is a useful strategy to provide increased Fusarium wilt resistance.

Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum

The necrotrophic fungal pathogen Fusarium pseudograminearum (F. pseudograminearum) causes crown rot disease (CR) in wheat. This host-pathogen interaction has not been studied previously at the molecular level. In this study. using real-time quantitative PCR, the expression of 26 selected wheat genes was examined 1, 2 and 4 days after inoculation of wheat seedlings of the CR susceptible cultivar Kennedy and the partially field-resistant cultivar Sunco. Reproducible induction of eight defence genes consisting of PR1.1, PR2 (beta,1-3 glucanase), PR3 (chitinase), PR4 (wheativin), PR5 (thaumatin-like protein). TaPERO (peroxidase), PR10 and TaGLP2a (germin-like) was observed. These genes were induced in both cultivars, however. some genes were induced more rapidly in Sunco than in Kennedy. MJ treatment also induced the above pathogen responsive defence genes in both cultivars while benzo(1,2,3)thiadiazole-7-carbothionic acid S-methyl ester (BTH) treatment weakly induced them in Kennedy only. Similarly. treatment with MJ before inoculation significantly delayed the development of necrotic symptoms for 2 weeks in both wheat cultivars, while BTH pre-treatments delayed symptom development in Kennedy only. The chemically induced protection, therefore, correlated with induction of the F. pseudograminearum-responsive genes. These results support the emerging role of jasmonate signalling in defence against necrotrophic fungal pathogens in monocots and future manipulation of this pathway may improve CR resistance in wheat. (c) 2006 Elsevier Ltd. All rights reserved.

Impacts of seawater acidification on mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation and energy metabolism, link to supplementary material

Marine organisms have to cope with increasing CO2 partial pressures and decreasing pH in the oceans. We elucidated the impacts of an 8-week acclimation period to four seawater pCO2 treatments (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 µatm) on mantle gene expression patterns in the blue mussel Mytilus edulis from the Baltic Sea. Based on the M. edulis mantle tissue transcriptome, the expression of several genes involved in metabolism, calcification and stress responses was assessed in the outer (marginal and pallial zone) and the inner mantle tissues (central zone) using quantitative real-time PCR. The expression of genes involved in energy and protein metabolism (F-ATPase, hexokinase and elongation factor alpha) was strongly affected by acclimation to moderately elevated CO2 partial pressures. Expression of a chitinase, potentially important for the calcification process, was strongly depressed (maximum ninefold), correlating with a linear decrease in shell growth observed in the experimental animals. Interestingly, shell matrix protein candidate genes were less affected by CO2 in both tissues. A compensatory process toward enhanced shell protection is indicated by a massive increase in the expression of tyrosinase, a gene involved in periostracum formation (maximum 220-fold). Using correlation matrices and a force-directed layout network graph, we were able to uncover possible underlying regulatory networks and the connections between different pathways, thereby providing a molecular basis of observed changes in animal physiology in response to ocean acidification.

Isolation and selection of plant growth-promoting bacteria associated with sugarcane

v. 46, n. 2, p. 149-158, apr./jun. 2016.

Secretome analysis of Trichoderma atroviride T17 biocontrol of Guignardia citricarpa

The fungal species Guignardia citricarpa is an important pathogen in citriculture. Members of the fungal genus Trichoderma are recognized as biocontrol agents but studies on the interactions between both fungi are scarce. This study aimed to identify extracellular proteins secreted by Trichoderma atroviride T17 that are related to the control of G. citricarpa. Two-dimensional gel electrophoresis (2D) was used to study the patterns of proteins secreted by T. atroviride T17 in medium containing glucose (control) and in medium containing G. citricarpa GC3 inactivated mycelium. We identified 59 of the 116 spots differentially expressed (50.86%) by LC–MS/MS. Of these, we highlight the presence of glycoside hydrolases (CAZy families 3, 43, 54, 76 and 93), chitinase, mutanase, a-1,3-glucanase, a-1,2-mannosidase, carboxylic hydrolase ester, carbohydrate-binding module family 13, glucan 1,3-b-glucosidase, a-galactosidase and Neutral protease 2. These proteins are related to mycoparasitism processes, stimuli and therefore to the biological control of pathogens. The results obtained are in agreement with reports describing an increase in the secretion of proteins related to mycoparasitism and biological control and a reduction in the secretion of proteins related to the metabolism of Trichoderma species grown in the presence of the pathogen. Moreover, these results are pioneer in understanding T. atroviride interaction with G. citricarpa. For the first time, we identified potential candidate proteins that may have a role in the antagonism mechanism of G. citricarpa by T. atroviride T17. Thus our results shed a light into the molecular mechanisms that T. atroviride use to control G. citricarpa.

Quitosana na indução de resistência ao tombamento de plântulas de espécies olerícolas e no controle de fitopatógenos in vitro

Induction of resistance is defined as the activation of a state of resistance against diseases which is induced systemically in plants by the use of biotic or abiotic agents without any modification of the plant genome, occurring non-specific way, by activating genes coding for various plant defense responses. Chitosan is a polymer derived from the deacetylation of chitin, which is found in large quantities in crustacean shell, and studied with the potential to control plant pathogens, both by its direct fungistatic action, as the ability to induce protection of plants, indicating the presence of molecules of elicitoras characteristics. Three experiments with objective of evaluating the potential of chitosan in the seedling resistance induction were developed, beet (Beta vulgaris) seeds, cucumber (Cucumis sativus) seeds and tomato (Solanum lycopersicum) seeds, and the control of Fusarium sp., Rhizoctonia solani K¨uhn e Pythium sp. in vitro conditions. The experimental design was completely randomized, with four replications. Beet seeds, tomato and cucumber were submerged in chitosan solution for 20 minutes, in concentrations of 0.25, 0.5, 1 and 2% in the control and distilled water. Seeds were sown in trays containing Plantmax Florestalr substrate sterilized and inoculated with Fusarium sp., Rhizoctonia solani K¨unh and Pythium sp., respectively for the three cultures. The experiment was conducted for 14 days in growth chamber with controlled temperature (25 C 2 C), light (12 hour photoperiod) and humidity (70% 10%). The evaluations were seed emergency, seedling damping-off, seedling length, fresh weight and activity of the enzymes phenylalanine amˆonia-liase (PAL), chitinase and b-1,3-glucanase. It was also rated the mycelial growth of Fusarium sp., Pythium sp. and R. solani on P.D.A. (Potato-Dextrose and Agar) culture medium containing chitosan at the same concentrations evaluated in seeds. For beet growing, seed treatment with chitosan presented higher emergence and the length of the seedlings, and reduced the percentage of tipping. Treatment with chitosan activated the systemic acquired resistance with expression of chitinase and b-1,3-glucanase enzymes. For the tomato crop in chitosan concentration of 0.25% favored the emergency of seedlings, reduced the incidence of tipping and activated the PAL enzymes, chitinase and b-1,3-glucanase. In cucumber on the concentration of up 0.5% favored seedlings emergence and reduces the incidence of tipping. Chitosan activated the PAL enzymes and b-1,3-glucanase. Chitosan also presented fungistatic action on the initial growth of Pythium sp. and R. solani in vitro conditions, however, such action did not prevail until the end of the experiment. To Fusarium sp. the concentration of chitosan resulted in the reduction of mycelial growth in vitro.

Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus , and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

Isolation and Identification of the Chitinolytic Bacteria from Rumen Ecosystem

Rumen is an interesting ecosystem for microbial exploration and their products. Isolation of the chitinolytic bacteria from the rumen ecosystem found 109 colonies that produced clear zone, 84 colonies (86%) anaerobic and 17 colonies (14%) aerobic. Clear zone appeared in the third and fourth days incubation. Four potential isolates were chosen for identification purposes. Results showed that the bacteria were sticky, gram-positive, motile, endospore-forming, mesophilic and aerobic. It was supposed to Bacillus spp. the optimal pH and temperature to produce chitinase from isolate 18 are pH 6.0 and temperature of 35-40ºC. Divalent cations Mg, Ca, Zn, and Mn increase chitinase activity, while Cu and Co inhibit enzyme activity. When isolate 18 was grown on shrimp waste meal, it showed aptimal activity on the fifth days incubation. (Animal Production 5(2): 73-78 (2003) Key Words : Isolation, Identification, Chitinolytic Bacteria, Rumen