Identification of RipAZ1 as an avirulence determinant of Ralstonia solanacearum in Solanum americanum

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease.     

Using both xylanase and laccase enzymes for pulp bleaching

Two enzyme treatments involving xylanase (X) and laccase (L) were used jointly in an XLE sequence (where E denotes alkaline extraction) to bleach oxygen-delignified eucalyptus kraft pulp in the presence of 1-hydroxybenzotriazol (HBT) as mediator. The results of the XLE sequence were compared with those of an LE sequence. The application conditions for the laccase–mediator system were optimized by using a sequential statistical plan involving three variables (viz., the laccase and mediator doses, and the reaction time) with both sequences. The models used to predict the kappa number and brightness revealed that, once all accessible lignin was removed, the system altered other coloured compounds. The best conditions for the L stage involved a reduced mediator dose (0.5% odp). The xylanase pretreatment increased the accessibility of residual lignin and facilitated removal of hexenuronic acids. For a specific target brightness level of 70% ISO, the X pretreatment can save as much as 30% laccase and 80% mediator while shortening the reaction time by 45%.     

Improved method for identifying and quantifying olive oil phenolic compounds and their metabolites in human plasma by microelution solid-phase extraction plate and liquid chromatography–tandem mass spectrometry

Two methods based on solid-phase extraction (SPE) using traditional cartridges and microelution SPE plates (μSPE) as the sample pre-treatment, and an improved liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) were developed and compared to determine the phenolic compounds in virgin oil olive from plasma samples. The phenolic compounds studied were hydroxytyrosol, tyrosol, homovanillic acid, p-coumaric acid, 3,4-DHPEA-EDA, p-HPEA-EDA, luteolin, apigenin, pinoresinol and acetoxypinoresinol. Good recoveries were obtained in both methods, and the LOQs and LODs were similar, in the range of low μM. The advantage of μSPE, in comparison with SPE cartridges, was the lack of the evaporation step to pre-concentrate the analytes. The μSPE-UPLC–ESI-MS/MS method developed was then applied to determine the phenolic compounds and their metabolites, in glucuronide, sulphate and methylated forms, in human plasma after the ingestion of virgin olive oil.     

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N_1-107) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N_1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co-evolution.     

Assessment of Genetic Stability Among In Vitro Plants of Arachis retusa Using RAPD and AFLP Markers for Germplasm Preservation

Arachis retusa Krapov. et W. C. Gregory et Valls is endemic in the West-central region of Brazil, occurring In areas endangered by human actions. The establishment of in vitro preservation methods for wild species of Arachis is an alternative to seed banks for germplasm storage, multiplication and distribution. The risk of genetic changes Induced by tissue culture and the monitoring of the genetic stability of the biological material before, during and after storage must be considered In the context of conservation. Random amplified polymorphlc ONA （RAPO） and amplified fragment length polymorphism （AFLP） fingerprinting were used to evaluate the genetic stability of in vitro plants originated from cotyledons and embryo axes of A. retusa. Cotyledons originated shoots through direct organogenesls and embryo axes displayed muItishoot formation Induced by 110 mmol/L and 8.8 mmol/L BAP, respectively. Ninety genomlc regions （loci） generated from RAPO and 372 from AFLP analyses were evaluated. All amplified fragments detected by both techniques in plants derived from the two explant types were monomorphic. The results Indicate that the recovered shoots are genetically stable at the assessed genomic regions.     

The role of BAP in somatic embryogenesis induction from seed explants of <Emphasis Type="Italic">Arachis</Emphasis> species from Sections <Emphasis Type="Italic">Erectoides</Emphasis> and <Emphasis Type="Italic">Procumbentes</Emphasis>

Somatic embryogenesis was induced from seed explants of Arachis archeri, A. porphyrocalix (Section Erectoides) and A. appressipila (Section Procumbentes) in response to 6-benzylaminopurine (BAP). Embryo axes first developed into single shoots in response to 4.4 μM BAP. Friable embryogenic calluses were produced from the hypocotyl region of these explants in response to different BAP concentrations. Embryonic leaflets also gave rise to friable calluses, but somatic embryos were only observed in explants of A. archeri and A. appressipila. Histological analyses revealed the presence of heart-shaped, torpedo and cotyledonary stages embryos, both as isolated and fused structures. A low frequency of embryo-to-plant conversion was achieved by inducing shoot development on medium solidified with 0.5% phytagel and supplemented with 1.5% or 3% sucrose. Rooting was induced on MS supplemented with indole-3-acetic acid (IAA).     

Packing features of the all-AT oligonucleotide d(AAATTT)

We describe the packing features of the oligonucleotide duplex d(AAATTT)2, as determined by X-ray diffraction. There is little information on sequences that only contain A and T bases. The present structure confirms that these sequences tend to pack as a helical arrangement of stacked oligonucleotides in a B conformation with Watson-Crick hydrogen bonding. Our results demonstrate that the virtual TA base step between stacked duplexes has a negative twist that improves base stacking. This observation is consistent with the low stability of TA base steps in B-form DNA.