Transposon Mutagenesis of Pseudomonas syringae Pathovars syringae and morsprunorum to Identify Genes Involved in Bacterial Canker Disease of Cherry

Bacterial canker of Prunus, affecting economically important stone fruit crops including cherry, peach, apricot and plum, is caused by the plant pathogen Pseudomonas syringae (P.s.). Strains from two pathovars—P.s. pv. syringae (Pss) and P.s. pv. morsprunorum race 1 (PsmR1) and 2 (PsmR2)—in three phylogenetically distant clades have convergently evolved to infect Prunus. The bacteria enter woody tissues through wounds and leaf scars, causing black necrotic cankers. Symptoms are also produced on blossom, fruit and leaves. Little is known about the mechanisms P.s. uses to colonise tree hosts such as Prunus. Here, we created transposon (Tn) mutant libraries in one strain of P.s. from each of the three clades and screened the mutants on immature cherry fruit to look for changes in virulence. Mutants (242) with either reduced or enhanced virulence were detected and further characterised by in vitro screens for biofilm formation, swarming ability, and pathogenicity on leaves and cut shoots. In total, 18 genes affecting virulence were selected, and these were involved in diverse functions including motility, type III secretion, membrane transport, amino acid synthesis, DNA repair and primary metabolism. Interestingly, mutation of the effector gene, hopAU1, led to an increase in virulence of Psm R2.

Download Full-text

Phage PPPL-1, a New Biological Agent to Control Bacterial Canker Caused by Pseudomonas syringae pv. actinidiae in Kiwifruit

Antibiotics ◽

10.3390/antibiotics10050554 ◽

2021 ◽

Vol 10 (5) ◽

pp. 554

Author(s):

Yu-Rim Song ◽

Nguyen Trung Vu ◽

Jungkum Park ◽

In Sun Hwang ◽

Hyeon-Ju Jeong ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Biocontrol Agent ◽

Control Agent ◽

Bacterial Canker ◽

Control Efficacy ◽

Antibiotic Resistant ◽

Canker Disease ◽

Recent Emergence ◽

Phage Cocktail

Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium that causes bacterial canker disease in kiwifruit. Copper or antibiotics have been used in orchards to control this disease, but the recent emergence of antibiotic-resistant Psa has called for the development of a new control agent. We previously reported that the bacteriophage (or phage) PPPL-1 showed antibacterial activity for both biovar 2 and 3 of Psa. To investigate the possibility of PPPL-1 to control bacterial canker in kiwifruit, we further tested the efficacy of PPPL-1 and its phage cocktail with two other phages on suppressing disease development under greenhouse conditions using 6 weeks old kiwifruit plants. Our results showed that the disease control efficacy of PPPL-1 treatment was statistically similar to those of phage cocktail treatment or AgrimycinTM, which contains streptomycin and oxytetracycline antibiotics as active ingredients. Moreover, PPPL-1 could successfully kill streptomycin-resistant Psa isolates, of which the treatment of BuramycinTM carrying only streptomycin as an active ingredient had no effect in vitro. The phage PPPL-1 was further characterized, and stability assays showed that the phage was stable in the field soil and at low temperature of 0 ± 2 °C. In addition, the phage could be scaled up quickly up to 1010 pfu/mL at 12 h later from initial multiplicity of infection of 0.000005. Our results indicate that PPPL-1 phage is a useful candidate as a biocontrol agent and could be a tool to control the bacterial canker in kiwifruit by Psa infection in the field conditions.

Download Full-text

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit

10.1101/2020.06.18.158634 ◽

2020 ◽

Author(s):

Jay Jayaraman ◽

Minsoo Yoon ◽

Emma R. Applegate ◽

Erin A. Stroud ◽

Matthew D. Templeton

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Close Relative ◽

Bacterial Canker ◽

In Planta ◽

Lesion Formation ◽

Canker Disease

SummaryPseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterised and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenised. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, likely due to the need to evade HopM1-triggered immunity in kiwifruit. We characterised the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.

Download Full-text

Transcriptome Analysis on the Mechanism of Ethylicin Inhibiting Pseudomonas syringae pv. actinidiae on Kiwifruit

Microorganisms ◽

10.3390/microorganisms9040724 ◽

2021 ◽

Vol 9 (4) ◽

pp. 724

Author(s):

Tao Liu ◽

Xiaoli Ren ◽

Guangyun Cao ◽

Xia Zhou ◽

Linhong Jin

Keyword(s):

Pseudomonas Syringae ◽

Biofilm Formation ◽

Protein Export ◽

Bacterial Canker ◽

Canker Disease ◽

Expression Of Genes ◽

Flagellar Assembly ◽

Plant Stem

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) is a devastating disease of kiwifruit, which is severely limiting the development of the kiwifruit industry. Ethylicin is a broad-spectrum plant biomimetic fungicide. However, its application in the control of kiwifruit bacterial canker is rarely reported, and the mechanism of ethylicin on Psa remains unknown. In this study, we investigated the effect of ethylicin on Psa in vitro and in vivo and found that ethylicin can inhibit the growth of Psa and prevent the cankering in the plant stem. Mechanism investigation indicated that ethylicin acted by limiting the movement of Psa, destroying the cell membrane of Psa, and inhibiting the formation of Psa biofilm. In addition, it was also found through transcriptomics research that ethylicin can up-regulate the expression of genes related to protein export and biofilm formation–Pseudomonas aeruginosa and down-regulate the expression of genes related to flagellar assembly in Psa. This study concluded that ethylicin can effectively inhibit Psa growth, and it could help to gain a better understanding of the mechanisms of ethylicin inhibiting Psa and provide practical data for the application of ethylicin as a highly potent agent for controlling the bacterial canker disease of kiwifruit.

Download Full-text

First Report of Bacterial Canker Caused by Pseudomonas syringae pv. morsprunorum Race 1 on Peach from Khyber Pakhtunkhwa Province of Pakistan

Plant Disease ◽

10.1094/pdis-10-17-1618-pdn ◽

2018 ◽

Vol 102 (10) ◽

pp. 2027-2027 ◽

Cited By ~ 2

Author(s):

R. Ahmed ◽

M. Inam-ul-Haq ◽

U. Shahzad ◽

S. Hyder ◽

S. Shahzaman ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Bacterial Canker ◽

First Report ◽

Khyber Pakhtunkhwa ◽

Race 1 ◽

Khyber Pakhtunkhwa Province

Download Full-text

Immunocytochemical Localization of HrpA and HrpZ Supports a Role for the Hrp Pilus in the Transfer of Effector Proteins from Pseudomonas syringae pv. tomato Across the Host Plant Cell Wall

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.3.394 ◽

2001 ◽

Vol 14 (3) ◽

pp. 394-404 ◽

Cited By ~ 58

Author(s):

Ian R. Brown ◽

John W. Mansfield ◽

Suvi Taira ◽

Elina Roine ◽

Martin Romantschuk

Keyword(s):

Cell Wall ◽

Pseudomonas Syringae ◽

Plant Cell ◽

Type Iii Secretion ◽

Plant Cell Wall ◽

Immunogold Labeling ◽

Effector Proteins ◽

Basal Bodies ◽

Type Iii

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 μm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Download Full-text

Relationships between kiwifruit bacterial canker disease and kiwifruit productivity

New Zealand Plant Protection ◽

10.30843/nzpp.2014.67.5719 ◽

2014 ◽

Vol 67 ◽

pp. 34-40 ◽

Cited By ~ 2

Author(s):

K.J. Froud ◽

N. Cogger ◽

R.M. Beresford

Keyword(s):

Pseudomonas Syringae ◽

Significant Relationship ◽

Regression Models ◽

Virulent Strain ◽

Bacterial Canker ◽

Infection Status ◽

Linear Regression Models ◽

Canker Disease ◽

Productivity Data ◽

The Relationship

Bacterial canker disease caused by a virulent strain of Pseudomonas syringae pv actinidiae (PsaV) has affected kiwifruit vines in New Zealand since 2010 This study investigated the association of PsaV with productivity within Hayward and Hort16A varieties PsaV infection status and date of diagnosis for 3309 infected orchards were provided by Kiwifruit Vine Health while Zespri provided productivity data Linear regression models were constructed to determine the relationship between production and PsaV infection in Hayward and Hort16A orchards Results showed a significant relationship between the numbers of weeks PsaV was detected in Hort16A orchards and a reduction in productivity This was likely due to the removal of Hort16A vines or productive areas of canopy in response to the presence of severe symptoms within an orchard A similar significant relationship was also found in Hayward orchards although the reduction in productivity was smaller and took longer to develop than in Hort16A

Download Full-text

The ecological genetics ofPseudomonas syringaefrom kiwifruit leaves

10.1101/235853 ◽

2017 ◽

Cited By ~ 1

Author(s):

Christina Straub ◽

Elena Colombi ◽

Li Li ◽

Hongwen Huang ◽

Matthew D. Templeton ◽

...

Keyword(s):

Population Structure ◽

Pseudomonas Syringae ◽

Bacterial Pathogen ◽

Ecological Factors ◽

Ecological Genetics ◽

Plant Interactions ◽

In Planta ◽

Canker Disease ◽

Pathogen Populations

SUMMARYInteractions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurringPseudomonas syringaeisolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused byP. syringaepv.actinidiae(Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenicPsaand commensalP. syringaePG3a isolated from the same leaf was tested using reciprocal invasion from rare assaysin vitroand in planta.P. syringaeG33C (PG3a) inhibitedPsaNZ54, while the presence ofPsaNZ54 enhanced the growth ofP. syringaeG33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system ofPsa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations.ORIGINALITY-SIGNIFICANT STATEMENTBacterial pathogen populations are often studied with little consideration of co-occurring microbes and yet interactions between pathogens and commensals can affect both population structure and disease progression. A fine-scale sampling of commensals present on kiwifruit leaves during an outbreak of bleeding canker disease caused byP. syringaepv.actinidiaereveals a clonal population structure. A new clade of non-pathogenicP. syringae(PG3a) appears to be associated with kiwifruit on a global scale. The presence of PG3a on kiwifruit has significant effects on the outcome of infection byP. syringaepv.actinidiae. This emphasises the value of studying the effect of co-occurring bacteria on pathogen-plant interactions.

Download Full-text

The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate–citrate exchange

The Plant Cell ◽

10.1093/plcell/koab223 ◽

2021 ◽

Cited By ~ 1

Author(s):

Chun Pong Lee ◽

Marlene Elsässer ◽

Philippe Fuchs ◽

Ricarda Fenske ◽

Markus Schwarzländer ◽

...

Keyword(s):

Systems Approach ◽

Acid Synthesis ◽

Amino Acid Synthesis ◽

Mitochondrial Carrier ◽

Cell Compartments ◽

Organic Acid Metabolism ◽

In Organello ◽

Mitochondrial Carrier Proteins

Abstract Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate–citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.

Download Full-text