The application of the CRISPR–Cas9 system in Pseudomonas syringae pv. actinidiae

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

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“…syringae pv. actinidiae ( Psa ) [ 50 ], Pseudomonas chlororaphis [ 51 ], Agrobacterium spp . [ 52 ], and Erwinia amylovora [ 53 ].…”

Section: Discussionmentioning

confidence: 99%

“…Besides Xoo, the CRISPR-mediated technologies for plasmid curing, gene knockout, and base editing have been preliminarily explored in only a few other bacterial plant pathogens, including Pst [25,49], P. syringae pv. actinidiae (Psa) [50], Pseudomonas chlororaphis [51], Agrobacterium spp. [52], and Erwinia amylovora [53].…”

Section: Plos Pathogensmentioning

confidence: 99%

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

et al. 2023

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“…This hypothesis is supported by a deletion of the NRPS gene in the P. syringae pv. actinidiae pyoverdine biosynthesis pathway using single-plasmid CRISPR-Cas9 resulting in a non-fluorescence phenotype [ 20 ]. This difference in phenotype complicates the identification process of P. syringae since P. syringae was originally described as a fluorescent phytopathogen [ 21 ] and this differential phenotype is still used in diagnostics today.…”

Section: Discussionmentioning

confidence: 99%

Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia

Djitro

Roach²,

Mann

et al. 2022

BMC Microbiol

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Background A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2. Results Average nucleotide identity refined the P. syringae clade 2b-a into four clusters (Cluster A, B, C1 and C2), an expansion from the previously identified A, B and C. Australian isolates were in Cluster A, C1 and C2. Genomic analyses highlighted several key factors that may contribute to the virulence of these isolates. Six orthologous groups, including three virulence factors, were associated with P. syringae phylogroup 2 cucurbit-infecting strains. A region of genome plasticity analysis identified a type VI secretion system pathway in clade 2b-a strains which could also contribute to virulence. Pathogenicity assays on isolates KL004-k1, KFR003-1 and 77-4C, as representative isolates of Cluster A, C1 and C2, respectively, determined that all three isolates can infect pumpkin, squash, watermelon and zucchini var. Eva with different levels of disease severity. Subsequently, type III effectors were investigated and four type III effectors (avrRpt2, hopZ5, hopC1 and hopH1) were associated with host range. The hopZ effector family was also predicted to be associated with disease severity. Conclusions This study refined the taxonomy of the P. syringae clade 2b-a, supported the association between effector profile and pathogenicity in cucurbits established in a previous study and provides new insight into important genomic features of these strains. This study also provided a detailed and comprehensive resource for future genomic and functional studies of these strains.

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“…Sequences were aligned using the GeneiousPrime alignment tool with default settings (v.2021.1.1) or via MAFFT using FFT-NS-2 (Katoh et al, 2005). The coffee-associated plasmid was cured from CB.PG4 isolates PgcUFLA48 and PgcUFLA116 and CB.PG3 isolates PtaUFLA142-1 and PtaUFLA142-2 by targeting the plasmid-localized avrD gene using the CRISPR-Cas9 system pJH1 (accession no: MN346704.1; Ho et al, 2020). The avrD targeting oligos used were 5 0 -AAACTAAAAGAAAGCCAA GGCTTGGACTCCCAAGG-3 0 and 5 0 -AAAACCTTGGGA GTCCAAGCCTTGGCTTTCTTTTA-3 0 .…”

Section: Plasmid Sequence Comparison and Curingmentioning

confidence: 99%

Pseudomonas syringae coffee blight is associated with the horizontal transfer of plasmid‐encoded type III effectors

McTavish,

Almeida,

Tersigni

et al. 2023

New Phytologist

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Summary The emergence of new pathogens is an ongoing threat to human health and agriculture. While zoonotic spillovers received considerable attention, the emergence of crop diseases is less well studied. Here, we identify genomic factors associated with the emergence of Pseudomonas syringae bacterial blight of coffee. Fifty‐three P. syringae strains from diseased Brazilian coffee plants were sequenced. Comparative and evolutionary analyses were used to identify loci associated with coffee blight. Growth and symptomology assays were performed to validate the findings. Coffee isolates clustered in three lineages, including primary phylogroups PG3 and PG4, and secondary phylogroup PG11. Genome‐wide association study of the primary PG strains identified 37 loci, including five effectors, most of which were encoded on a plasmid unique to the PG3 and PG4 coffee strains. Evolutionary analyses support the emergence of coffee blight in PG4 when the coffee‐associated plasmid and associated effectors derived from a divergent plasmid carried by strains associated with other hosts. This plasmid was only recently transferred into PG3. Natural diversity and CRISPR‐Cas9 plasmid curing were used to show that strains with the coffee‐associated plasmid grow to higher densities and cause more severe disease symptoms in coffee. This work identifies possible evolutionary mechanisms underlying the emergence of a new lineage of coffee pathogens.

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The application of the CRISPR–Cas9 system in Pseudomonas syringae pv. actinidiae

Cited by 6 publications

References 53 publications

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia

Pseudomonas syringae coffee blight is associated with the horizontal transfer of plasmid‐encoded type III effectors

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