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Additional file 3. Confirmation of the presence of plasmids for RNAi-mediated silencing of Pc-yap1 and Pc-rsmA and analysis of silencing in P. chrysogenum transformants. (A) Close-up of the pGpdPki-RNAi vector region with the opposite-oriented gpd and pki promoters, and the plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA with the inserted DNA fragments from the Pc-yap1 and Pc-rsmA genes at the NcoI site. Primers Gpd1(pki1)R and Pki1(gpd1)F are shown at the position of annealing with sequences in the gpd and pki promoters, respectively. The expected size of amplified DNA fragments in each type of transformant is indicated with double-headed arrows. (B) Agarose gel with the result of PCR amplification with primers Gpd1(pki1)R and Pki1(gpd1)F using as template DNA from the purified pGpdPki-RNAi vector (lane V), DNA from strain C1 containing the pGpdPki-RNAi vector (lane C1) and DNA from strain Wis54-1255 (lane W). (C) Results of the PCR amplification performed with the mentioned primers and DNA from eight transformants with the pGpdPki-RNAi/PcYap1 plasmid (Y1…. Y18). (D) Results of the PCR amplification performed with the mentioned primers and DNA from eight transformants with the pGpdPki-RNAi/PcRsmA plasmid (R24… R39). (E) Silencing of expression of Pc-yap1 (upper panels) and Pc-rsmA (lower panels) in selected strains containing the RNAi-silencing plasmids pGpdPki-RNAi/PcYap1 and pGpdPki-RNAi/PcRsmA, respectively. RNA was extracted from mycelium grown for 60 h in MPPY medium and used for semiquantitative RT-PCR (as described by Domínguez-Santos et al. [96]) using primers qPcYap1-F and -R for analysis of Pc-yap1 expression, qRsmA-F and -R for Pc-rsmA, and qactA-F and -R for act. The left panels show the intensity of the bands in an agarose gel loaded with the products of the RT-PCR reactions, and the right panels the densitometry analysis of the bands. The results were normalized with the bands of the constitutively expressed act gene. The parental strain Wis54-1255 (lane W) was used as reference with a value for integrated optical density (IOD) of 100. Y8: strain si-PcYap1-8; Y12: si-PcYap1-12; R24: si-PcRsmA-24; R25: si-PcRsmA-25.

Additional file 2. Recombinant PCR for overexpression of genes Pc-yap1 and Pc-rsmA. The pki gene promoter from A. niger was fused to fragments from the genes extending from the ATG start codon to around 300 bp downstream the TGA stop codon to ensure the presence of the transcriptional terminator. The position of the primers used for PCR reactions is indicated (see Materials and Methods for details). The final fragments with the genes fused to the pki promoter were digested with the restriction enzymes EcoRV and SpeI (Ppki::Pc-yap1) or KpnI and XhoI (Ppki::Pc-rsmA) and inserted in the vector pBKSpyrG to obtain the plasmids pPyrG-pki::Pc-yap1 and pPyrG-pki::Pc-rsmA, respectively; the restriction sites in the primers are highlighted in color. Introns are indicated in grey color. Added restriction sites at the 5’-end of the primers are highlighted in orange color.

Additional file 8. Clustal W alignment of PcYap1 with S. cerevisiae Yap1 and S. pombe Pap1, and conservation of functional domains between them. Nuclear import and export sequences (NLS and NES) are highly conserved. Also conserved are the cysteine-rich domains N-CRD and C-CRD, the latter overlapping the NES, and the key cysteine residues forming disulphide bonds described in Yap1 which mask the NES and prevent export form the nucleus, resulting in nuclear location of the protein in oxidative conditions (see text for details).

Additional file 7. EMSA to analyze possible interactions between PcYap1 and PcRsmA upon binding to their respective binding sites. Probes containing either the PcYap1-binding site (PTA1-WT), the PcRsmA binding site (RsmA-2C) or both (upPta1) were incubated with their respective binding proteins (lanes 2, 4, 6, 7) or with both proteins (lanes 3, 5, 8). The results show that no apparent interaction between the proteins occurs when one of them recognizes and binds its specific site (lanes 3, 5). In lane 8 a supershift takes place. Probe upPta1 contains both binding sites for each of the proteins, therefore the supershift pattern does not demonstrate interaction between the proteins and can be explained by the simultaneous binding of the proteins to their respective sites; nevertheless, interaction when this simultaneous binding occurs cannot be excluded.

Additional file 1. Strategy for silencing of expression of Pc-yap1 and Pc-rsmA. (A) A DNA fragment of 458 bp from the exon 2 of Pc-yap1 was amplified by PCR with primers siYAP1-F and -R, digested with NcoI and inserted at the NcoI site of plasmid pGpdPki-RNAi to obtain plasmid pGpdPki-RNAi/PcYap1. (B) A DNA fragment of 409 bp from the exon 3 of the Pc-rsmA gene was amplified by PCR with primers siRsmA-F and -R, digested with NcoI and inserted at the NcoI site of plasmid pGpdPki-RNAi to obtain plasmid pGpdPki-RNAi/PcRsmA. (C) Strategy of silencing. Transcription from the opposite-oriented promoters gpd and pki generates complementary RNA strands that form a dsRNA with the sequence of the inserted 458 bp fragment from Pc-yap1. This dsRNA will cause silencing of the expression of Pc-yap1 through the RNAi pathway.

Additional file 5. Isolation of Pc-yap1, Pc-rsmA and Pc-atf21 CDS and insertion in expression vectors for P. pastoris. (A) An RT-PCR was performed using RNA from a submerged culture and primers Yap-1 and Yap-6, which contain restriction sites for KpnI and ApaI, respectively. The amplified fragment was inserted in the pPICZ-A vector after digestion with these enzymes and ligation. The in-frame insertion is shown, indicating the position of the cloning enzymes, the Pc-yap1 CDS and the c-myc-6xHis tag. (B) For Pc-rsmA, a recombinant PCR approach was followed, designing primers with sequences linking exon 1 to 2 (RsmA-2 and RsmA-3) and exon 2 to 3 (RsmA-4 and RsmA-5), plus primer RsmA-1, annealing at the start of the ORF and containing an EcoRI restriction site at 5’, and primer RsmA-6, annealing at the end of the ORF and containing an XbaI restriction site. PCR reactions were performed to separately amplify exon 1 (primers RsmA-1 and -2), exon 2 (primers RsmA-3 and -4) and exon 3 (primers RsmA-5 and -6). The products of the first two reactions were mixed together and a new PCR was performed with primers RsmA-1 and -4 to fuse exons 1 and 2. Finally, the product of the latter reaction was mixed with the product of the reaction of exon 3 and a PCR was performed with primers RsmA-1 and RsmA-6 to obtain the entire CDS, from the ATG to the last codon (next to the Stop codon but not including it). This fragment was digested with the enzymes EcoRI and XbaI and inserted in the pPICZ-B vector digested with the same enzymes, so that the Pc-rsmA gene ORF was fused in frame with the sequences in the vector encoding the c-myc epitope and the 6xHis tag. (C) For Pc-atf21, the previously cloned gene was used as template for amplification with primers AtfB-1 and AtfB-2, since this gene contains no introns. The cloning procedure in vector pPICZ-B was as described above. (Asterisks indicate additional amino acids in the fusion proteins resulting from the cloning strategies. In the case of Pc-atf21, the cloning strategy resulted in the loss of the last amino acid, Asn).

Additional file 6. Purification of recombinant c-myc-6xHis-tagged PcYap1, PcRsmA and PcAtf21 proteins after expression in P. pastoris. Protein extracts were obtained as described in Materials and Methods and loaded onto Ni-NTA Spin 50 (Qiagen) columns. The tagged proteins were eluted with increasing concentrations of imidazole. Aliquots of the eluted samples were analyzed by SDS-PAGE, lane M: size marker, lane CE: protein crude extract, imidazole concentrations were 100, 250, 300 and 500 mM for PcYap1::c-myc-6xHis, and 50, 200, 300 and 500 mM for PcRsmA::c-myc-6xHis and PcAtf21::c-myc-6xHis. Bottom right panel, partially purified PcRsmA::c-myc-6xHis and PcAtf21::c-myc-6xHis run on the same gel for size comparison purposes.

Additional file 4. Confirmation of the presence of plasmids for overexpression of Pc-yap1 and Pc-rsmA in P. chrysogenum transformants. (A) Scheme of the fusion of the pki promoter with the Pc-yap1 (top) and Pc-rsmA (down) genes at the ATG start codon in the plasmids pPyrG-pki::Pc-yap1 and pPyrG-pki::Pc-rsmA. Primers Pki1(gpd1)F, OE-PcYap1-R, siYAP1-R, OE-RsmA-R and siRSMA-R are shown at the position of annealing with sequences in the pki promoter and the Pc-yap1 and Pc-rsmA genes, respectively. The expected size of amplified DNA fragments in each type of transformant for every primer pair is indicated with double-headed arrows. (B) Agarose gel with the result of PCR amplification with primers Pki1(gpd1)F and OE-PcYap1-R using as template DNA from the purified pPyrG-pki::Pc-yap1 plasmid (lane P), DNA from strain Wis54-1255 (lane W), a transformant with the empty pBKSpyrG vector (lane Y) and transformants OE::PcYap1-A through –F (lanes A through F). Transformants selected for further characterization are indicated with a red circle. (C) Result of PCR amplification with primers Pki1(gpd1)F and OE-RsmA-R using as template DNA from transformants OE::PcRsmA-A through –H (lanes A through H). (D) Result of PCR amplification with primers Pki1(gpd1)F and siRSMA-R using as template DNA from the purified pPyrG-pki::Pc-rsmA plasmid (lane P), total DNA from strain Wis54-1255 (lane W), a transformant with the empty pBKSpyrG vector (lane Y) and transformants OE::Pc-rsmA-C through –H (lanes C through H). Transformants selected for further characterization are indicated with a red circle.

Additional file 9. Clustal W alignment of PcRsmA with A. nidulans RsmA, C. albicans FCR3, and S. cerevisiae Yap3. An NLS is well conserved in all proteins at the same position as in the Yap1 proteins (the beginning of the bZIP domain). Three putative NES were detected with LocNES (from Support Vector Machine) in the amino acid sequence of PcRsmA; two of them, NES-2 and NES-3, show some conservation with the equivalent regions in the other three proteins. NES-3 is located at the C-terminus and between the only two cysteine residues present in PcRsmA, which should form a disulphide bond in oxidative conditions that would cause the conformational change detected by SDS-PAGE (Fig. 7). A cysteine rich domain located at the N-terminus in Yap3 is not conserved in any of the other proteins.

Philaenus spumarius (Ps) is the main xylem-sap feeder vector of Xylella fastidiosa pauca ST53 (XF), the bacterium responsible for the OQDS symptoms, a devastating plant disease now invading the olive orchards of Apulia. Vector control is expected to be the main action to manage insect-borne pathogens and to contain the following diseases. We suggest tuning the actual IPM (Integrated Pest Management) strategy to manage the transmission events instead of the vector only. Achievements in Aphrophoridae sampling techniques permit to add a physical (mechanical) control action against vectors eggs in IPM as hitherto sketched. A series of subsequent control actions imposed into a restricted time interval decimate juveniles. Only adults acquire XF feeding on infected plants, to infect (primarily), re-infect and super-infect (secondarily) without latency the olives they encounter until the recipient plants hard too much to be accepted. We consider vector IPM during pre-imaginal life for overall population control and adult control in the way we suggest both crucial to prevent and protect the plants from the infections. The lattice model we present can help to shape IPM by forecasting the effectiveness of adult control actions through the analysis of the way in which the transmission depends on 1) spacing; 2) number, frequency, intensity, and kind of control actions, and 3) vector mobility in open field or greenhouse-like environments.