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Background: Plasma AR status has been identified as a potential biomarker of response in metastatic castration-resistant prostate cancer (mCRPC) patients receiving docetaxel or AR-targeted therapies. However, the relevance of plasma AR in the overall management of CRPC patients receiving different docetaxel doses is unknown., Patients and Methods: This was a multi-institution study of associations between baseline plasma AR copy number status, assessed by droplet digital PCR, and outcome in 325 mCRPC patients receiving docetaxel at standard or adapted regimen at the discretion of the treating physician. Upon analysis, patients were assigned randomly to either a training (n = 217) or validation (n = 108) cohort., Results: In the training cohort, AR-gained patients treated with adapted docetaxel regimen had a significantly worse median progression-free survival (PFS) (3.8 vs 6.3 months, hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.34-4.95, p < 0.0001), median overall survival (10.8 vs 20.6 months, HR 1.98, 95% CI 1.09-3.62, p = 0.0064) and PSA response (PSA > -50%: odds ratio 4.88 95%CI 1.55-14.32, p = 0.013) as compared to plasma AR normal patients. These findings were all confirmed in the validation cohort. However, in patients treated with standard docetaxel regimen, these differences were not seen. The interaction between AR CN status and dose reduction of docetaxel was considered as independent factor for PFS in both the training and validation cohort (HR 2.84, 95% CI 1.41-5.73, p = 0.003, and HR 4.79, 95% CI 1.79-12.82, p = 0.002)., Conclusion: Despite the retrospective non-randomised design of this study, our hypothesis-generating findings could suggest plasma AR as a potential biomarker for optimal docetaxel timing and dose in mCRPC patients. Prospective trials are warranted., Competing Interests: Conflict of interest statement G. Attard certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (e.g., employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: G. Attard reports receiving commercial research grants from Janssen, Arno Therapeutics, and Innocrin Pharma; has received honoraria and/or travel support from the speakers' bureaus of Janssen, Astellas, Sanofi-Aventis, and Roche/Ventana; has received travel support from Pfizer, Abbott Laboratories, Bayer Healthcare, and Essa Pharmaceuticals; has ownership interest (including patents) in the Institute of Cancer Research Rewards to Inventors; and is a consultant for/advisory board member of Janssen-Cilag, Veridex, Bayer Healthcare, Roche/Ventana, Astellas, Medivation, Pfizer, Novartis, Millennium Pharma, Abbott Laboratories, and Essa Pharma. V. Conteduca, E. Gonzalez-Billalabeitia, and U. De Giorgi received speaker honoraria or travel support from Astellas, Janssen-Cilag, and Sanofi-Aventis. V. Conteduca received consulting fee from Bayer and is an advisory board member of Janssen. E. Castro reports receiving commercial research grants from AstraZeneca, Bayer, Janssen; has received honoraria and/or travel support from the speakers’ bureaus of Astra Zeneca, Astellas, Bayer, Janssen, Pfizer, Bristol Myers Squibb and Roche; and is an advisory board member of Astellas, Bayer and Janssen. N. Romero Laorden has received honoraria and/or travel support from Bayer, Astellas, Janssen-Cilag, and Sanofi-Aventis. D. Olmos has a compensated advisory role for Astellas, AstraZeneca, Bayer, Clovis, Genetech/Roche, Janssen, and uncompensated advisory role for BioOncotech, Tokai; has received a speaker fee from Astellas, Bayer, Janssen, Sanofi, and travel support from Astellas, Bayer, Janssen, Roche; has received research funding (to the institution): AstraZeneca, BioOncotech, Bayer, Janssen. M. Marín-Aguilera has received travel support from Bristol Myers Squibb. Aventis. B. Mellado reports receiving commercial research grants from Roche and Bayer; has received travel support from Pfizer and Janssen; and is a consultant for/advisory board member of Roche, Sanofi, Janssen, Astellas Oncology, Pfizer, Novartis, Bristol Myers Squibb and Ipsen. No potential conflicts of interest were disclosed by other authors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The rhizobacterium Pseudomonas alcaligenes AVO110 exhibits antagonism toward the phytopathogenic fungus Rosellinia necatrix . This strain efficiently colonizes R. necatrix hyphae and is able to feed on their exudates. Here, we report the complete genome sequence of P. alcaligenes AVO110. The phylogeny of all available P. alcaligenes genomes separates environmental isolates, including AVO110, from those obtained from infected human blood and oyster tissues, which cluster together with Pseudomonas otitidis . Core and pan-genome analyses showed that P. alcaligenes strains encode highly heterogenic gene pools, with the AVO110 genome encoding the largest and most exclusive variable region (~1.6 Mb, 1795 genes). The AVO110 singletons include a wide repertoire of genes related to biofilm formation, several of which are transcriptionally modulated by R. necatrix exudates. One of these genes ( cmpA ) encodes a GGDEF/EAL domain protein specific to Pseudomonas spp. strains isolated primarily from the rhizosphere of diverse plants, but also from soil and water samples. We also show that CmpA has a role in biofilm formation and that the integrity of its EAL domain is involved in this function. This study contributes to a better understanding of the niche-specific adaptations and lifestyles of P. alcaligenes , including the mycophagous behavior of strain AVO110.

Background: Intraductal (IDC) and cribriform (CRIB) histologies in prostate cancer have been associated with germline BRCA2 (gBRCA2) mutations in small retrospective series, leading to the recommendation of genetic testing for patients with IDC in the primary tumour., Patients and Methods: To examine the association of gBRCA2 mutations and other tumour molecular features with IDC and/or cribriform (CRIB) histologies, we conducted a case-control study in which primary prostate tumours from 58 gBRCA2 carriers were matched (1:2) by Gleason Grade Group and specimen type to 116 non-carriers. Presence/absence of IDC and CRIB morphologies was established by two expert uropathologists blinded to gBRCA2 status. Fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS) were used to detect BRCA2 alterations, PTEN deletions and TMPRSS2-ERG fusions. Chi-squared tests were used to compare the frequency of IDC and CRIB in gBRCA2 carriers and controls and to assess associations with other variables. Logistic regression models were constructed to identify independent factors associated with both histology patterns., Results: No significant differences between gBRCA2 carriers and non-carriers were observed in the prevalence of IDC (36% gBRCA2 versus 50% non-carriers, p = 0.085) or CRIB (53% gBRCA2 versus 43% non-carriers p = 0.197) patterns. However, IDC histology was independently associated with bi-allelic BRCA2 alterations (OR 4.3, 95%CI 1.1-16.2) and PTEN homozygous loss (OR 5.2, 95%CI 2.1-13.1). CRIB morphology was also independently associated with bi-allelic BRCA2 alterations (OR 5.6, 95%CI 1.7-19.3)., Conclusions: While we found no association between gBRCA2 mutations and IDC or CRIB histologies, bi-allelic BRCA2 loss in primary prostate tumours was significantly associated with both variant morphologies, independently of other clinical-pathologic factors., Competing Interests: Conflict of interest statement R.L. declares speaker fees from Roche, Janssen, Sanofi and Bayer, and travel support from Roche, Janssen, Sanofi and Astellas Pharma. S.S. declares honoraria from Amgen, Bristol-Meyers Squibb, Merck, Merck Serono consulting or Advisory Role from AstraZeneca and Merck, research funding from AstraZeneca, Merck Sharp and Dohme, Amgen and Endocyte. F.L-C. declares consulting or Advisory Role from Astella Pharma, speaker fees from Janseen, Astellas Pharma, Research Funding from Astellas Pharma (Inst) and travel support from Astellas Pharma and Janssen. J.R-B. declares consulting or Advisory Role from Janssen, Astellas Pharma and Bayer, and Research Funding from HealthMDx (Inst). J.R.B declares consulting or advisory role from Astellas Pharma, Bayer and Janssen-Cilag and research funding from MDxHealth (Inst). C.L. declares honoraria from Roche and travel support from Astellas Pharma and Angelini. N.R-L. declares speaker fees from MSD, consulting or Advisory Role from Ipsen, Astellas Pharma, Bayer, Tesaro, AstraZeneca and Sanofi, research funding from Janssen (Inst), and Pfizer (Inst) and travel support from Janssen. F.Z. declares expert testimony for Sanofi and travel support from Ipsen. D.L. declares speaker fees from Janssen, Bayer, Astellas Pharma, Sanofi, Pfizer and BMS, consulting or Advisory Role from Sanofi, and travel support from Janssen, and Astellas Pharma. J.M. declares consulting or advisory role from Astra Zeneca, Janssen and Roche, speaker's bureau from Astellas Pharma, AstraZeneca and Sanofi and travel support from AstraZeneca, Ipsen and Sanofi. C.C.P. declares consulting or Advisory Role from AstraZeneca and Promega. E.S.A. declares honoraria and consulting or advisory role from Astellas, AstraZeneca, Clovis Oncology, Dendreon, ESSA, Janssen Biotech, Medivation, Merck, Sanofi; Research Funding from Aragon Pharmaceuticals (Inst), Astellas (Inst), AstraZeneca (Inst), Clovis Oncology (Inst), Constellation Pharmaceuticals (Inst), Dendreon (Inst), Exelixis (Inst), Genetech (Inst), Janssen Biotech (Inst), Johnson&Johnson (Inst), Merck (Inst), Millennium (Inst), Novartis (Inst), Sanofi (Inst) and Tokai pharmaceuticals (Inst); co-inventor of a biomarker technology licensed to Qiagen. D.O. declares honoraria from Bayer, Janssen and Sanofi, consulting or Advisory Role from AstraZeneca, Bayer, Clovis, Daiichi-Sankyo, Janssen, MSD and Roche, research funding from Astellas (Inst), AstraZeneca (Inst), Bayer (Inst), Genentech (Inst), Janssen (Inst), Medivation (Inst), MSD (Inst), Pfizer (Inst), F. Hoffman-Roche (Inst) and Tokai Pharmaceutics (Inst) and travel support from Bayer, Ipsen, Janssen and Roche. T.L. declares consulting or advisory role for Janssen and research funding from DeepBio and Ventana Medical Systems. E.C. declares honoraria from Astellas Pharma, AstraZeneca, Bayer, Clovis, Janssen, Pfizer and Roche, consulting or Advisory Roles for AstraZeneca, Bayer, Janssen, MSD and Pfizer, research funding from AstraZeneca (Inst), Bayer (Inst) and Janssen (Inst) and travel support from Astra Zeneca, Bayer and Janssen. D.C.S, I.M.A., H.T., A.M.G-P., L.M., T.D., A.S., J.D.P, I.G., M.I.P., T.G. and P.P-L. do not have any relationships to disclose., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Over the past years, several studies have demonstrated that defects in DNA damage response and repair (DDR) genes are present in a significant proportion of patients with prostate cancer. These alterations, particularly mutations in BRCA2, are known to be associated with an increased risk of developing prostate cancer and more aggressive forms of the disease. There is growing evidence that certain DDR gene aberrations confer sensitivity to poly-(ADP ribose) polymerase inhibitors and/or platinum chemotherapy, while other defects might identify cases that are more likely to benefit from immune checkpoint inhibition. The potential prognostic impact and relevance for treatment selection together with the decreasing costs and broader accessibility to next-generation sequencing have already resulted in the increased frequency of genetic profiling of prostate tumours. Remarkably, almost half of all DDR genetic defects can occur in the germline, and prostate cancer patients identified as mutation carriers, as well as their families, will require appropriate genetic counselling. In this review, we summarise the current knowledge regarding the biology and clinical implications of DDR defects in prostate cancer, and outline how this evidence is prompting a change in the treatment landscape of the disease.

Background: A common polymorphism (1245A>C) in the HSD3B1 gene is associated with increased de novo synthesis of androgens and worse outcomes in men treated with androgen-deprivation therapy for metastatic castration-sensitive prostate cancer. The objective of the study was to determine whether this polymorphism is associated with outcomes for metastatic castration-resistant prostate cancer (mCRPC) treated with abiraterone or enzalutamide., Patients and Methods: A total of 547 patients treated with abiraterone or enzalutamide from two prospective cohorts were evaluated. The HSD3B1 genotype was determined by targeted sequencing and/or TaqMan single-nucleotide polymorphism genotyping. In cohort 1, patients were randomized to receive abiraterone + prednisone or enzalutamide. In cohort 2, patients received either agent according to investigator's choice. Prostate-specific antigen (PSA) response rate, time to PSA progression (TTPP), time to progression (TTP) and overall survival were determined. Associations between HSD3B1 genotypes and outcomes were evaluated via univariate Cox regression. Multivariable Cox model was used to determine the independent association of each covariate., Results: The HSD3B1 variant genotype (CC) was present in 15% of patients and was associated with worse TTP [hazard ratio (HR) 1.31, 95% confidence interval (CI) 1.02-1.67, P = 0.032] and PSA response rates (48% for CC versus 62% and 65% for AA and AC, respectively [P = 0.019]), with no significant difference in TTPP (HR 1.28, 95% CI 0.99-1.66, P = 0.064). The effect of genotype was similar for treatment with abiraterone or enzalutamide with a negative test for interaction for TTPP (P = 0.997) and TTP (P = 0.749). Multivariable analysis did not show a significant association between genotype and TTP or TTPP., Conclusions: The HSD3B1 (CC) genotype was associated with shorter TTP and lower PSA response rate in patients with mCRPC treated with abiraterone or enzalutamide. However, the CC genotype did not provide prognostic information beyond that conferred by standard clinical variables, suggesting that it may not be a suitable stand-alone biomarker in mCRPC., Competing Interests: Disclosures DJK reports consulting fees from Bayer. RL reports consulting or advisory role from Sanofi, Aventis; Speakers' Bureau from Janssen-Cilag, Roche; Research Funding from Janssen-Cilag (Inst), Bayer (Inst) and Travel, Accommodations from Janssen-Cilag, Roche. DL reports consulting or advisory role from Janssen Oncology, Sanofi, Bayer Health, Speakers' Bureau from Janssen Oncology, Bayer Health, Astellas Pharma, Sanofi and Travel, Accommodations and Expenses from Janssen Oncology, Sanofi, Astellas, Pharma, Celgene. DLF reports consulting fees from Janssen, Astellas and Bayer. NR-L reports honoraria from Bayer, Pfizer, Astellas, Sanofi, Janssen, Roche, MSD; research funding from Janssen-Cilag (Inst), Bayer (Inst), Astellas Pharma (Inst), Sanofi (Inst) and travel and accommodations from Pfizer, Janssen-Cilag, Roche. JV reports honoraria from Pfizer, Janssen, Bayer, Bristol-Myers Squibb and Astellas and travel and consulting fees from Astellas, Merck and Bristol-Myers Squibb. MEG reports patents OGX-011 and OGX-427. AWW reports grants and personal fees from Janssen and honorarium from Bayer, AstraZeneca and Janssen. DO reports honoraria from Bayer, Janssen, Sanofi; consulting or advisory role from Janssen, Bayer, AstraZeneca, Clovis Oncology; research funding from AstraZeneca (Inst), Bayer (Inst), Janssen (Inst), Genentech (Inst), Roche (Inst), Pfizer (Inst), Astellas Medivation (Inst), Tokai Pharmaceuticals (Inst) and travel, accommodations and expenses from Bayer, Janssen, Ipsen. KNC reports grants from Janssen and Astella, Sanofi, AstraZeneca, Bayer, Pfizer and Roche; and personal fees from Janssen, Astellas, Sanofi, AstraZeneca, Bayer, Pfizer and Roche. EC reports honoraria from Astellas Pharma, Janssen-Cilag, AstraZeneca, Bayer, consulting or Advisory Role from Bayer, Janssen, Bayer (I), Janssen (I), research funding from AstraZeneca (Inst), Bayer (Inst), Janssen (Inst) and travel, accommodations and expenses from Bayer, Janssen, Roche, Astellas Pharma. IMA, MA, ST, YC, CO, MIP and MZ have no relationships to disclose., (Copyright © 2020 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Human myogenic precursor cells have been isolated and expanded from a number of skeletal muscles, but alternative donor biopsy sites must be sought after in diseases where muscle damage is widespread. Biopsy sites must be relatively accessible, and the biopsied muscle dispensable. Here, we aimed to histologically characterize the cremaster muscle with regard number of satellite cells and regenerative fibres, and to isolate and characterize human cremaster muscle-derived stem/precursor cells in adult male donors with the objective of characterizing this muscle as a novel source of myogenic precursor cells. Cremaster muscle biopsies (or adjacent non-muscle tissue for negative controls; N = 19) were taken from male patients undergoing routine surgery for urogenital pathology. Myosphere cultures were derived and tested for their in vitro and in vivo myogenic differentiation and muscle regeneration capacities. Cremaster-derived myogenic precursor cells were maintained by myosphere culture and efficiently differentiated to myotubes in adhesion culture. Upon transplantation to an immunocompromised mouse model of cardiotoxin-induced acute muscle damage, human cremaster-derived myogenic precursor cells survived to the transplants and contributed to muscle regeneration. These precursors are a good candidate for cell therapy approaches of skeletal muscle. Due to their location and developmental origin, we propose that they might be best suited for regeneration of the rhabdosphincter in patients undergoing stress urinary incontinence after radical prostatectomy.

Stress urinary incontinence (SUI) affects 200 million people worldwide. Standard therapies often provide symptomatic relief, but without targeting the underlying etiology, and show tremendous patient-to-patient variability, limited success and complications associated with the procedures. We review in this article the latest clinical trials performed to treat SUI using cell-based therapies. These therapies, despite typically including only a small number of patients and short term evaluation of results, have proven to be feasible and safe. However, there is not yet a consensus for the best cell source to be used to treat SUI and not all patients may be suitable for these therapies. Therefore, more clinical trials should be promoted recruiting large number of patients and evaluating long term results., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Context: The urinary tract, previously considered a sterile body niche, has emerged as the host of an array of bacteria in healthy individuals, revolutionizing the urology research field., Objective: To review the literature on microbiome implications in the urinary tract and the usefulness of probiotics/prebiotics and diet as treatment for urologic disorders., Evidence Acquisition: A systematic review was conducted using PubMed and Medline from inception until July 2016. The initial search identified 1419 studies and 89 were included in this systematic review., Evidence Synthesis: Specific bacterial communities have been found in the healthy urinary tract. Changes in this microbiome have been observed in certain urologic disorders such as urinary incontinence, urologic cancers, interstitial cystitis, neurogenic bladder dysfunction, sexually transmitted infections, and chronic prostatitis/chronic pelvic pain syndrome. The role of probiotics, prebiotics, and diet as treatment or preventive agents for urologic disorders requires further investigation., Conclusions: There is a microbiome associated with the healthy urinary tract that can change in urologic disorders. This represents a propitious context to identify new diagnostic, prognostic, and predictive microbiome-based biomarkers that could be used in clinical urology practice. In addition, probiotics, prebiotics, and diet modifications appear to represent an opportunity to regulate the urinary microbiome., Patient Summary: We review the urinary microbiome of healthy individuals and its changes in relation to urinary disorders. The question to resolve is how we can modulate the microbiome to improve urinary tract health., (Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Surgical treatment of diseases affecting long urethral areas represents a challenge in urology. Recent developments of tissue-engineered urethral substitutes represent a hope for patients. However finding an ideal tissue source for urethral reconstruction first requires proper understanding of the native human urethra physiology and a deep knowledge of the histological and molecular features of the native human urethra. Here we present a comprehensive characterization of male and female urethra by histological, histochemical and immunohistochemical methods with a panel of 15 antibodies. The results demonstrated that the histology of the male and female urethra depend on the area where the sample is taken along its length. Proximal areas of male and female urethra have differential expression of the epithelial basal and suprabasal layer markers CK14 and CK10 which distinguished the prostatic/membranous and proximal female urethra from the bulbar/penile and distal female areas of the urethra. The distal male (penile) and female may be further divided by the distinct expression pattern of CK19. On the other hand, the expression of CK5/6 and CK19 also make a distinction of the proximal and distal female urethra. These results should facilitate a more informed selection of donor graft tissues for urethral replacement. Besides, novel bioengineered urethral tissue approaches should take into account the characterization of the different areas of the urethra presented in this work.

Introduction: Diabetes and cardiovascular disease are risk factors for erectile dysfunction (ED). Selective inhibitors of the type 5 phosphodiesterase are the first option for treating ED. However, it is unknown why there are patients with low response to this treatment. Polymorphisms in the PDE5A gene may influence the response to PDE5 inhibitors treatment., Aim: The aim of this study is to analyze the relationship between PDE5A polymorphisms, diabetes, and the efficacy of sildenafil treatment., Methods: A Spanish prospective cohort of 170 Caucasian male patients diagnosed with ED and ischemic heart disease treated with angioplasty was studied., Main Outcome Measures: ED was evaluated according to the 5-item version of the International Index for Erectile Function before and after treatment with sildenafil 50 mg. The gene sequence of the PDE5A gene was analyzed for the presence of rs12646525 and rs3806808 polymorphisms. Glucose and glycosylated hemoglobin levels were measured in blood serum samples. The relationship between treatment response, genotype, and glycemic status was analyzed., Results: Patients with G-allele of rs3806808 polymorphism showed a worse response to the treatment compared to TT-homozygote patients. Nondiabetic G-allele carriers showed a worse treatment response than TT-homozygotes patients. These differences were not seen in diabetic patients. There were no significant differences in treatment response according to the rs12646525 polymorphism in total population or according to the glycemic status. Logistic regression analysis showed that nondiabetic carriers of the major allele of both the rs12646525 and rs3806808 polymorphism had a significantly higher likelihood to respond to the treatment than diabetic patients carriers of the minor allele (P < .05)., Conclusion: The response to sildenafil treatment depends on polymorphisms in the PDE5A gene and the glycemic status of the patients., (Copyright © 2016 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear., Results: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants., Conclusions: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c-di-GMP) phosphodiesterase-encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free-living and virulence-related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour-inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real-time monitoring of olive plants infected with green fluorescent protein (GFP)-tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild-type strain. All free-living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c-di-GMP signalling in the virulence of this group of plant pathogens., (© 2014 BSPP AND JOHN WILEY & SONS LTD.)

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

Despite a recent burst of research, knowledge on c-di-GMP signaling pathways remains largely fragmentary and molecular mechanisms of regulation and even c-di-GMP targets are yet unknown for most bacteria. Besides genomics or bioinformatics, accompanying alternative approaches are necessary to reveal c-di-GMP regulation in bacteria with complex lifestyles. We have approached this study by artificially altering the c-di-GMP economy of diverse pathogenic and mutualistic plant-interacting bacteria and examining the effects on the interaction with their respective host plants. Phytopathogenic Pseudomonas and symbiotic Rhizobium strains with enhanced levels of intracellular c-di-GMP displayed common free-living responses: reduction of motility, increased production of extracellular polysaccharides and enhanced biofilm formation. Regarding the interaction with the host plants, P. savastanoi pv. savastanoi cells containing high c-di-GMP levels formed larger knots on olive plants which, however, displayed reduced necrosis. In contrast, development of disease symptoms in P. syringae-tomato or P. syringae-bean interactions did not seem significantly affected by high c-di-GMP. On the other hand, increasing c-di-GMP levels in symbiotic R. etli and R. leguminosarum strains favoured the early stages of the interaction since enhanced adhesion to plant roots, but decreased symbiotic efficiency as plant growth and nitrogen contents were reduced. Our results remark the importance of c-di-GMP economy for plant-interacting bacteria and show the usefulness of our approach to reveal particular stages during plant-bacteria associations which are sensitive to changes in c-di-GMP levels.

Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century witnessed a large number of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively prevented the pathogen from benefitting from the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A number of studies in recent years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other nonpathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, which encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi-olive interaction and to obtain insights into why some pathovars like it necrotic and why some like it knot., Synonyms: Pseudomonas syringae pv. savastanoi., Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi., Microbiological Properties: Gram-negative rods, 0.4-0.8 × 1.0-3.0 μm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25-30 °C; oxidase negative, arginine dihydrolase negative; elicits the hypersensitive response on tobacco; most isolates are fluorescent and levan negative, although some isolates are nonfluorescent and levan positive., Host Range: P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive have been reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii are differentiated from pathovar savastanoi mostly in their host range, and were not formally recognized until 1996. Literature before about 1996 generally names strains of the three pathovars as P. syringae ssp. savastanoi or P. savastanoi ssp. savastanoi, contributing to confusion on the host range and biological properties., Disease Symptoms: Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits., Epidemiology: The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated with nonpathogenic bacteria, both epiphytically and endophytically, and have been demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans, which could result in increased bacterial populations and disease symptoms., Disease Control: Based on preventive measures, mostly sanitary and cultural practices. Integrated control programmes benefit from regular applications of copper formulations, which should be maintained for at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen., Useful Websites: http://www.pseudomonas-syringae.org/; http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)