The elicitin secreted by Phytophthora palmivora, a rubber tree pathogen

Palmivorein, a new member of the elicitin family, was purified from the culture filtrate of Phytophthora palmivora isolated from the rubber tree, Hevea brasiliensis. The elicitin was obtained by ammonium sulfate precipitation and further purified using ion-exchange and gel filtration. The molecular weight, isoelectric point, amino acid composition and N-terminal sequences of this molecule are reported and compared to other known elicitins. Palmivorein, as determined by SDS-PAGE, is a small protein of M(r) ca. 10,000. It is classified as an alpha-elicitin according to its acidic pI and the valine residue at position 13. Like other elicitins, the P. palmivora elicitin causes tissue necrosis on tested tobacco leaves. It also causes severe wilting and necrosis of Hevea tissue, and leaves of the susceptible rubber clone (with respect to P. palmivora) are much more sensitive to this elicitin than those that are resistant.     

Expression of a novel mycobacterial phosphodiesterase successfully lowers cAMP levels resulting in reduced tolerance to cell wall–targeting antimicrobials

cAMP and antimicrobial susceptibility in mycobacteriaAntimicrobial tolerance, the ability to survive exposure to antimicrobials via transient nonspecific means, promotes the development of antimicrobial resistance (AMR). The study of the molecular mechanisms that result in antimicrobial tolerance is therefore essential for the understanding of AMR. In gram-negative bacteria, the second messenger molecule 3′’,5′’-cAMP has been previously shown to be involved in AMR. In mycobacteria, however, the role of cAMP in antimicrobial tolerance has been difficult to probe due to its particular complexity. In order to address this difficulty, here, through unbiased biochemical approaches consisting in the fractionation of clear protein lysate from a mycobacterial strain deleted for the known cAMP phosphodiesterase (Rv0805c) combined with mass spectrometry techniques, we identified a novel cyclic nucleotide-degrading phosphodiesterase enzyme (Rv1339) and developed a system to significantly decrease intracellular cAMP levels through plasmid expression of Rv1339 using the constitutive expression system, pVV16. In Mycobacterium smegmatis mc²155, we demonstrate that recombinant expression of Rv1339 reduced cAMP levels threefold and resulted in altered gene expression, impaired bioenergetics, and a disruption in peptidoglycan biosynthesis leading to decreased tolerance to antimicrobials that target cell wall synthesis such as ethambutol, D-cycloserine, and vancomycin. This work increases our understanding of the role of cAMP in mycobacterial antimicrobial tolerance, and our observations suggest that nucleotide signaling may represent a new target for the development of antimicrobial therapies.     

Biosynthesis of scopoletin in Hevea brasiliensis leaves inoculated with Phytophthora palmivora

Inoculation of resistant (R) and susceptible (S) Hevea brasiliensisleaves with Phytophthora palmivorainduced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Heveaphytoalexin. The degree of resistance of four clones, as classified by the necrotic lesions, was related to the rapidity and intensity of Scp production. The resistant BPM-24, and marked partially resistant clone PB-235, displayed an early secretion of scopoletin that intensified and lasted longer than the weak partially resistant RRIT251, and susceptible clone RRIM600. The lesion size and amount of Scp after infection were positively correlated to the concentration of spores applied to Hevealeaves. In addition, in leaflets inoculated with high spore concentration, Scp reached the highest level earlier than those with low spore concentration. A fungitoxic effect of Scp on mycelium growth was shown in bioassays; the I₅₀ value tested on Phytophthora palmivora was relatively the same as on Phytophthora botryosa, but much lower than those found on other leaf pathogens of rubber tree, Corynespora cassiicolaand Colletotrichum gloeosporioides. The lesion size and level of Scp upon spore inoculation may be appropriate for classifying Heveaclones according to their R/S with respect to P. palmivora     

Purification of a Protease Inhibitor from Hevea brasiliensis cell suspension and it’s effect on the growth of Phytophthora palmivora

Protease inhibitors (PIs) are one family of pathogenesis-related proteins (PR-proteins) that play essential roles in defense mechanisms against an attack by a pathogenic microorganism or insect. Cell suspension derived from a seed integument of rubber tree (Hevea brasiliensis) treated for 48 h with 20 μM copper sulphate, an abiotic elicitor, had an increased production of PIs. The intracellular PIs were detected in an extract of treated cells; however, much higher levels of PIs were found in the medium (extracellular). Using azocasein as substrate, these PIs possessed strong inhibitory activity against subtilisin A but not against trypsin, chymotrysin and papain. These extracellular PIs were purified by anion exchange chromatography, DEAE-Sepharose (CL-6B), eluted with 0.06 M NaCl in 20 mM Tris-HCl (pH 7.0). The active fractions were then subjected to native and SDS preparative gel electrophoresis, respectively. A single band of a purified PI with a molecular weight of 25 kDa was revealed after a tricine SDS-PAGE and stained with silver nitrate. The yield of this purified protein was 3.14 ng.g^?1. The activity of the purified PI was stable up to 70 °C, and its activity was retained in the buffer pH values of 2–10. The biological activity of the obtained PI was investigated. It was found that the PI at 5 μg.mL^?1 (0.2 μM) inhibited the mycelium growth of Phytophthora palmivora, a rubber tree pathogen.