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All PCRs were done by using the Pfx polymerase (Invitrogen), and DNA fragments obtained were recombined into the p DONR201 vector by using the Gateway system. Standard procedures for yeast growth and yeast transformation were used (25).
The 5-fluoroorotic acid (5-FOA) selective plates contained minimal medium containing yeast nitrogen base without amino acids (Difco) and glucose, supplemented with lysine, leucine, uracil, and 1 mg/ml 5-FOA. Molecular biology techniques were performed by using standard protocols unless otherwise noted (26, 27). A gene and its downstream sequences was PCR-cloned with the following primers: 5′-TGG TAC CAC ACG ACT GAT CGT GCT T-3′ and 5′-TGA ATT CTG GTC AAG AAG TCC TTC C-3′.
This vector was generated by the insertion of the d III site of p GR0029 (29).
The N-terminal fusions with GFP were generated by using the Gateway-compatible p AM-PAT-GFP vector (a gift from F. Transient Transformation of Arabidopsis Protoplasts. Protoplasts were prepared from an Col-0 cell suspension culture (30).
NB-LRR proteins fall into two subclasses based on their N-terminal motifs.
One group possesses an N-terminal coiled-coil domain, whereas the second subclass shares similarities to the cytoplasmic Toll IL-1 receptor (TIR) domains of human and , the causal agent of bacterial wilt (21).
We verified that the PMT1 signal corresponded only to the GFP emission and the PMT2 signal only to the RFP emission.
For each construct, at least 10 different transformed 50 genes encoding candidate TTSS-dependent effectors, including several proteins homologous to Avr proteins described in other bacterial plant pathogens (32). To identify the Avr determinant recognized by RRS1-R, a set of disruption mutants generated in strain GMI1000 were screened for their pathogenicity on gene encodes a 52.8-k Da protein (National Center for Biotechnology Information accession no.
All of the subsequent steps were performed as described (31).
After a 36-h incubation, transformed protoplasts were observed by using a confocal microscope (Leica Microsystems, Heidelberg).
The laser settings were the following: 488 nm at 37% of maximal power and 543 nm at 100% of maximal power.
According to the guard model (9, 12), such effectors can associate and induce modifications of plant targets functioning as negative regulators of basal defense responses leading to disease development in plants lacking the corresponding R protein.
In a resistant host, the plant target that interacts with both R and Avr proteins is guarded by the R protein, preventing its manipulation by pathogen effectors.