Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonas tolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonas agarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.

dehydrogenase gene (RS23005) on one flank thus showing a similar feature as the quinone-IagB linkage in P. tolaasii. A gene for a putative SipB effector was also found to be part of this gene cluster. Only three other potential effector genes were identified in the genome: HopJ (RS03325), HrpK (RS18850) and HopAC1 (RS07770), with similarity to P. gingeri (all >90% identity). Neither cluster 1 or 3 had obvious T3SS transcriptional regulators associated with them nor were potential HrpL promoter sequences identified in the genome except for hrpK and hopAC1.
Searches for a T6SS identified three large gene clusters: RS02645-02770 was arranged in two operons, in opposing directions, with the RS02710-02770 cluster being flanked by large %GC drops; RS21080-21155 was arranged in two operons, in opposing directions, with an Rhs gene on one side (RS21150); RS21510-21580 was arranged as one operon and next to an 8kb region (RS21475-21505) containing a transposase, resolvase and integrase. The entire region is flanked by low %GC ratio drops. A smaller cluster was found at RS12550-12585 and flanked by low %GC ratio regions. A potential type VI-secreted lysozyme-like gene (RS09100) was found. Five genes annotated as Hcp1 Type VI effectors (RS01160, RS02725, RS09880, RS19150, RS19520) were identified as well as four Rhs genes (RS01355, RS05175, RS13620, RS20175) and several pseudogenes (Supplementary Material Table 5).

Pseudomonas sp. NS1
BLASTp analysis of the different secretion systems indicated P. fluorescens as the closest hits. Screening of the genome sequence identified two T3SS gene clusters, from RS10335-10445 (23 genes) and RS25340-25440 (21 genes). Large drops in the %GC ratio were observed flanking both regions. A putative tRNA-Met (RS10330) was observed on the left flank of the first gene cluster. Notably, two genes (RS10325-10320) were observed on the other side of the tRNA-Met, a putative phenazine biosynthesis protein, PhzF, and ggt. Together there is some syntenic similarity to P. tolaasii. The T3SS genes appeared to be organised into two to four operons with gene matches most closely matching P. fluorescens F113. A putative RspL (HrpL) gene (RS10335) with 97% match was found on the left flank of the cluster next to the tRNA-Met and a putative sigma-54 transcriptional regulator (RS10445) with a 77% match (amino acids) to RspR was on the right flank. A potential RspL (HrpL) promoter was found upstream of RS10340 (hrpJ) and RS10440.
The RS25340-25440 cluster consisted of 21 genes with genes with two potential operons one strand separated by a tetratricopeptide repeat protein gene (RS25410) on the opposing strand. No obvious transcriptional regulators or RspL promoters were observed. On the right flank, after the low %GC region, were three genes encoding putative luxR regulator, superoxide dismutase and peroxidase, showing a synteny similar to P. tolaasii.
Also similar to P. tolaasii was the discovery of an orphan hrcC gene (RS29245) contiguous with downstream genes (four instead of three), a HP, transcriptional regulator, iagB and quinonedependent dihydroorotate dehydrogenase genes.
Searches for a T6SS identified three large gene clusters: RS05740-05865 was arranged in two operons, in opposing directions with a low %GC region between them; RS18005-18080 was arranged as one operon flanked by low %GC ratio regions; RS24120-24190 was arranged as one operon, except RS24190 in the opposing direction, flanked by low %GC ratio regions. Two single Hcp1 Type VI effector (RS18030 and RS24190) and VgrG (RS08970 and RS17060) were identified with high similarity to P. fluorescens (>90%).

Primer
Sequence