Next Article in Journal
Investigation of Potent Antifungal Metabolites from Marine Streptomyces bacillaris STR2 (MK045300) from Western Algeria
Previous Article in Journal
Allelic Variation in gtfB–gtfC Region of Natural Variant of Streptococcus mutans Without Biofilm Formation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Bacteria
Volume 3
Issue 4
10.3390/bacteria3040026
bacteria-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Analysis of Infection Strategies of Pseudomonas cannabina pv. alisalensis and P. syringae pv. tomato in Different Host Plants

by
Nanami Sakata
1,2,*,
Giyu Usuki
1,
Kanon Yamamoto
1 and
Yasuhiro Ishiga
1
1
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Ibaraki, Japan
2
Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, Tsushima-naka 1-1-1, Kita-ku, Okayama 700-8530, Okayama, Japan
*
Author to whom correspondence should be addressed.
Bacteria 2024, 3(4), 379-389; https://doi.org/10.3390/bacteria3040026
Submission received: 16 September 2024 / Revised: 10 October 2024 / Accepted: 5 November 2024 / Published: 7 November 2024
Browse Figures
Versions Notes

Abstract

:
Plant pathogenic Pseudomonas species cause a variety of diseases in plants. Each Pseudomonas species employs different virulence factors and strategies for successful infection. Moreover, even the same bacterial pathogens can differentially utilize virulence factors against various host plants. However, there has been relatively less emphasis on comparing the infection strategies of a single bacterial pathogen on different hosts and different bacterial pathogens on a single host. Here, we investigated plant–pathogen interactions using two Pseudomonas species, Pseudomonas cannabina pv. alisalensis (Pcal) KB211 and Pseudomonas syringae pv. tomato (Pst) DC3000, and their host plants, cabbage and tomato. Our findings reveal distinct behaviors and virulence patterns across different host plants. Pcal multiplies to greater levels in cabbage compared to tomato, suggesting that Pcal is more adaptive in cabbage than tomato. Conversely, Pst showed robust multiplication in tomato even at lesser inoculum levels, indicating its aggressiveness in the apoplastic space. Gene expression analyses indicate that these pathogens utilize distinct virulence-related gene expression profiles depending on the host plant. These insights highlight the importance of revealing the spatiotemporal regulation mechanisms of virulence factors.

1. Introduction

Plant pathogenic Pseudomonas causes several disease symptoms, including blight, cankers, leaf spots, and galls on different host plants [1]. The disease cycle of foliar plant pathogenic Pseudomonas species is divided into two phases: the epiphytic phase, when bacteria survive on the leaf surface and penetrate through natural opening sites such as stomata, and the endophytic phase, when bacteria multiply in the plant apoplastic space and cause visible disease-associated symptoms [2]. One of the well-studied plant pathogenic Pseudomonas, P. syingae pv. tomato (Pst) DC3000 is a highly aggressive pathogen once inside host tissues by using a wide range of effector repertories, while it is a relatively weak epiphyte [2]. Pst DC3000 was originally isolated from tomato but can cause disease on the model plant Arabidopsis thaliana [2]. P. cannabina pv. alisalensis (Pcal) has a wide host range including dicot and monocot plants [3,4]. Although there is no clear evidence regarding whether Pcal shows a preference for the epiphytic or endophytic phase, initial Pcal entry into cabbage correlates with disease severity and maximum bacterial populations [5]. Pcal KB211 can infect a wide range of Brassicaceae plants, as well as tomato [3]. So far, there has been relatively less emphasis on comparing the infection strategies of different bacterial pathogens on a single host. Therefore, we aimed to compare the infection strategies of Pcal and Pst on cabbage and tomato.
Both Pcal and Pst use multiple virulence factors to successfully infect their hosts. One of the most important virulence factors is the type-three secretion system (T3SS), which derivers proteins known as type-three effectors (T3Es). Effector repertoires vary in size and composition among Pseudomonas strains [6]. The induction of T3SS genes is regulated by HrpL, an alternative sigma factor recognizing the hrp box in the promoter of T3SS genes. Further, some plant pathogenic bacteria produce phytotoxins that injure plant cells and affect symptoms or disease development. P. syringae produces several phytotoxins, including coronatine (COR), tabtoxin, syringolin, and phaseolotoxin [7], with both Pcal KB211 and Pst DC3000 producing COR [8,9,10]. COR is a hybrid molecule consisting of coronafacic acid (CFA) and coronamic acid (CMA) [11]. The sigma factor CorR positively regulates CFA and CMA biosynthesis [12]. Other sigma factors such as AlgU are also important in supporting P. syringae growth and disease development [13,14,15], while PvdS is involved in iron acquisition and contributes to virulence [16,17]. Further, the flagellin gene fliC is highly conserved because it is essential in bacterial motility. However, some pathogens change parts of the flagellin protein, called flg22 epitopes, to avoid detection by the plant immune system [18,19]. These epitopes are normally recognized by the plant receptor FLS2. In the case of Pcal, its flg22 epitopes are altered so that they are not recognized by FLS2 in A. thaliana [18]. Each Pseudomonas species employs different virulence factors and has a different strategy for successful infection.
We previously conducted a screen for Pcal mutants with reduced virulence on cabbage and identified Pcal potential virulence factors [20]. Some virulence factors we identified were required for both cabbage and oat infection, but others were not required for oat infection [20]. COR contributes to bacterial multiplication inside plants but might function differentially for Pcal virulence in cabbage and oat [8]. Elizabeth and Bender (2007) also demonstrated that COR contributes to Pst DC3000 bacterial multiplication differentially in host collard and turnip [21]. Thus, it is tempting to speculate that even the same bacterial pathogens can use virulence factors differentially against different host plants.
Therefore, we aimed to understand bacterial pathogen behavior during infection using two different host plants and two different bacterial pathogens. Pcal can infect both cabbage and tomato, and tomato is a host plant for the well-studied Pst DC3000. Therefore, we focused on comparing Pcal and Pst in tomato, as well as comparing Pcal behavior in cabbage and tomato. Our results showed that Pcal is a more adaptive pathogen on cabbage than on tomato. The virulence-related gene expression profiles also showed different patterns during infection on different host plants. Pst is a highly aggressive pathogen once in planta. The Pst expression patterns were also different from that of Pcal. Our results imply the ingenious strategies of each bacterial pathogen for successful infection of their host plants.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

The bacterial strains utilized in this study are listed in Supplementary Table S1. These include the Pseudomonas cannabina pv. alisalensis strain (Pcal) KB211 WT, T3SS mutant (NB35), COR mutant (ΔcmaA), and flagellar motility mutant (ΔfliC). Additionally, Pseudomonas syringae pv. tomato (Pst) DC3000 WT, the T3SS mutant (hrcC mutant), the COR mutant (DB29), and the flagellar motility mutant (ΔfliC) were used. All strains were cultured on King’s B (KB; [22]) medium at 28℃. Pcal NB35 was cultured on KB medium with 50 µg/mL kanamycin. Pst DB29 was cultured on KB medium with rifampicin at 100 μg/mL, kanamycin at 25μg/mL, and spectinomycin at 25 μg/mL. Pst hrcC mutant was cultured on KB medium with chloramphenicol at 25 μg/mL. Prior to inoculation, bacterial suspensions were prepared in sterile distilled water, and their cell densities at 600 nm (OD600) were determined using a Biowave CO8000 Cell Density Meter (Funakoshi, Tokyo, Japan). An OD600 of 0.1 corresponds to approximately 5 × 107 colony forming units (CFU/mL).

2.2. Plant Materials and Growth Conditions

For virulence assays with Pcal, cabbage (Brassica oleracea var. capitata) cv. Kinkei 201 was used, while tomato (Solanum lycopersicum) cv. Moneymaker was used for Pcal and Pst virulence assays. Plants were germinated from seed and grown under controlled conditions (23-25°C with a light intensity of 200 μEm-2s-1 and a 16 h light/8 h dark photoperiod). Cabbage and tomato plants were used in syringe inoculation at around three and four weeks post-germination, respectively.

2.3. Bacterial Inoculation Methods

To assess disease development, plants were syringe-inoculated with bacterial suspensions of varying concentrations (5 × 103, 5 × 104, 5 × 105, and 5 × 106 CFU/mL) into leaves using a 1 mL blunt syringe. The plants were then maintained at 70-80% relative humidity throughout the experiment. Bacterial populations in the plant were quantified at 0, 1, 2, 3, 4, and 5 days post-inoculation (dpi). Leaves were removed and photographed at 3 dpi.
To measure bacterial growth, leaf discs were collected from inoculated areas using a 3.5 mm-diameter cork-borer. The samples were homogenized, serially diluted in sterile distilled water, and plated onto solid KB agar. The bacterial colony forming units (CFUs) were counted after 2-3 days of incubation and normalized to CFU per cm2, using the leaf square centimeters. These bacterial growth assays were repeated in at least three independent experiments. Growth rate was calculated with the following equation: μ= {log (CFU/cm2) (t) – log (CFU/cm2) (0)}/ t.

2.4. Monitoring Bacterial Gene Expressions in Planta

To study gene expression during infection, Pcal at 5 × 103 and 5 × 105 CFU/mL was syringe-infiltrated into cabbage and tomato leaves. Similarly, tomato plants were syringe-infiltrated with Pst at the same bacterial concentrations. Total RNA was extracted from cabbage and tomato leaves at 2, 3, and 4 dpi using RNAiso Plus (Takara Bio, Kusatsu, Japan), followed by purification. RNA extraction and real-time quantitative RT-PCR (RT-qPCR) were performed as previously described [23]. Two micrograms of total RNA were treated with gDNA Remover (Toyobo, Osaka, Japan) to eliminate genomic DNA and then reverse transcribed using the ReverTra Ace qPCR RT Master Mix (Toyobo). The resulting cDNA was diluted 1:10 and used in RT-qPCR with THUNDERBIRD SYBR qPCR Mix (Toyobo) and the specific primers (listed in Supplementary Table S2) on a Thermal Cycler Dice Real-Time System (Takara Bio). Pcal KB211 outer membrane porin F (oprF) and recombinase A (recA) were used to normalize Pcal gene expression. Pst DC3000 outer membrane lipoprotein precursor (oprI), pyrroline-5-carboxylate reductase (proC), and RNA polymerase sigma factor (rpoD) were used to normalize Pst gene expression. Reagent blank (no-template) controls were used to detect contamination. The expression profiles were evaluated in at least six independent samples.

2.5. Statistical Analysis

All data are presented as mean ± standard error (SE). Statistical analyses were carried out using RStudio (version 1.6.0). Tukey’s honestly significant difference (HSD) test was used to analyze data. Differences of p < 0.05 were considered statistically significant.

3. Results

3.1. Pcal Multiplication in Cabbage and Tomato

We first investigated the Pcal population in host cabbage and tomato plants. When we inoculated cabbage with Pcal, cabbage inoculated with 5 × 103 CFU/mL showed no necrosis and fewer chlorosis symptoms compared to that with 5 × 105 and 5 × 106 CFU/mL but maintained great bacterial populations at 5 dpi (Figure 1A,C,E,F). In tomato inoculated with Pcal, bacterial populations reached only 6.58 log (CFU/cm2) (Figure 1C–F), while the maximum population in cabbage was 7.67 log (CFU/cm2) (Figure 1F). Although tomato inoculated with 5 × 103 CFU/mL did not show symptoms, Pcal maintained great bacterial populations (Figure 1B,C). To compare the Pcal growth rate in cabbage and tomato, we calculated the growth rate from 0 to 2 dpi (Table 1). The growth rates in cabbage were higher than that in tomato, indicating that Pcal grows well in cabbage. Together, these results suggest that Pcal might be a better colonizer in cabbage than in tomato.

3.2. Gene Expression Profiles of Pcal During Infection in Cabbage and Tomato

Since Pcal multiplied differently in host cabbage and tomato, we next investigated Pcal virulence-related gene expression profiles during infection in both plants. When we inoculated cabbage and tomato with Pcal at 5 × 103 CFU/mL inoculum levels, virulence-related gene expression was not significantly different (Figure 2A–I). When we inoculated cabbage at the 5 × 105 CFU/mL inoculum levels, the expression of all tested genes, including T3SS-related genes (hrpL, hopM1, and avrPto), COR-related genes (corR, cfl, andcmaA), pvdS, algU, and fliC, were upregulated at 2 dpi compared to that during tomato infection (Figure 2A–I). The expression of T3SS-related genes, COR-related genes, and pvdS were greater in tomato than in cabbage at 4 dpi (Figure 2A–I). Since Pcal could not multiply from 1 dpi in tomato when we inoculated at 5 × 105 CFU/mL (Figure 1D), these results suggest that the upregulation of virulence-related genes at the early infection stage is important for successful multiplication. The expression of algU was reduced continuously during Pcal tomato infection (Figure 2H). Furthermore, fliC expression during tomato infection was significantly lower than during cabbage infection at 2 dpi (Figure 2I). These results indicate that even the same bacterial pathogen, Pcal, exhibits different gene expression profiles depending on the host plant.

3.3. Pst Multiplication in Tomato

We next investigated the Pseudomonas syringae pv. tomato (Pst) population during tomato infection. While tomato inoculated with 5 × 103 CFU/mL inoculum levels showed fewer disease symptoms compared to that with 5 × 106 CFU/mL at 5 dpi (Figure 3A), the bacterial populations in tomato inoculated with 5 × 103 CFU/mL inoculum levels were greater than with 5 × 106 CFU/mL inoculum levels at 3 dpi (Figure 3B). To compare the growth rates, we selected the bacterial populations of Pcal (Figure 1D) and Pst (Figure 3B) in tomato and calculated the growth rate using the results at 5 × 103 and 5 × 105 CFU/mL by dividing the proliferation number by the proliferation number on the previous day (Supplementary Figure S2). While Pcal growth in cabbage initially increased in an inoculum-dependent manner by 3 dpi (Figure 1B), Pst populations in tomato inoculated at 5 × 104 CFU/mL were not significantly different compared to those in tomato inoculated at 5 × 105 and 5 × 106 CFU/mL at 2 dpi (Figure 3B).

3.4. Pst Gene Expression Profiles During Infection in Tomato

We also investigated Pst virulence-related gene expression during infection in tomato. Expression of T3SS-related genes (including hrpL, avrE1, and hopM1), COR-related genes (including corR, cfl, and cmaB), algU, and pvdS were significantly greater when inoculated at 5 × 105 CFU/mL, while the gene expression was stable when we inoculated at 5 × 103 CFU/mL (Figure 4A–H). Conversely, fliC expression was greater when we inoculated at 5 × 103 CFU/mL (Figure 4I).

3.5. Disease Phenotypes and Bacterial Multiplication of Pcal and Pst T3SS, COR, and Flagellar Motility Mutants

Virulence-related genes were expressed differentially depending on the inoculum levels (Figure 2). Therefore, to investigate the virulence factor roles in different concentrations, we inoculated cabbage with a Pcal T3SS mutant (NB35), COR mutant (ΔcmaA), and flagellar motility mutant (ΔfliC) at different inoculum concentrations. The bacterial populations of the T3SS mutant were significantly less than that of WT Pcal when we inoculated at 5 × 103 CFU/mL and 5 × 105 CFU/mL (Figure 5B,C). These results indicate that the T3SS is important for bacterial growth inside plants. Moreover, ΔfliC populations were significantly reduced compared to WT (Figure 5B,C). Conversely, there were no significant differences in the bacterial populations among WT Pcal and these mutants when we inoculated at 5 × 107 CFU/mL (Figure 5D), indicating that these virulence factors were not required for bacterial growth after it reached sufficient bacterial populations. However, leaves inoculated with NB35 showed no symptoms at 5 × 107 CFU/mL (Figure 5A). The bacterial populations of ΔcmaA were not significantly different compared to that of WT at 3 dpi regardless of the inoculum level (Figure 5B–D).
We next conducted the inoculation test using Pst and host tomato. We inoculated tomato with Pst WT, a T3SS mutant (hrcC mutant), a COR mutant (DB29), and a flagellar motility mutant (ΔfliC). The hrcC mutant populations were significantly less than those of WT Pst when we inoculated at 5 × 103 CFU/mL, indicating that the T3SS is important for Pst multiplication in tomato only at lesser cell density (Figure 5F). While Pcal ΔfliC showed reduced virulence when we inoculated at 5 × 103 and 5 × 104 CFU/mL, the Pst ΔfliC populations showed no significant differences or increased with WT (Figure 5F,G). Further, same as Pcal, there were no significant differences in the bacterial populations among WT and these mutants when we inoculated at 5 × 107 CFU/mL (Figure 5H). Similarly to cabbage inoculated with Pcal, tomato leaves inoculated with the hrcC mutant showed no symptoms at 5 × 107 CFU/mL (Figure 5E). The bacterial populations of DB29 were also not significantly different or greater compared to those of WT regardless of the inoculum levels (Figure 5F–H).

4. Discussion

We compared Pcal behavior across its two host plants, cabbage and tomato. Additionally, we contrasted the infection behaviors of two Pseudomonas strains, Pcal and Pst, on tomato. Our results suggest that Pcal could be better adapted to infect cabbage than tomato. Moreover, our results demonstrated that the initial bacterial densities were not significantly correlated with infection severity, suggesting that Pst is a highly aggressive pathogen once in the apoplastic space. This study underscores the importance of understanding the diverse infection strategies of various Pseudomonas species and their interactions with different host plants.
Our results raise several conceptual issues that are worth discussing. First, we demonstrated that Pcal multiplied to greater levels in cabbage than in tomato (Figure 1). Moreover, when we inoculated plants at 5 × 105 CFU/mL inoculum levels, Pcal virulence-related genes showed greater expression during infection in cabbage than in tomato at 2 dpi (Figure 2). These results suggest that Pcal is more adapted to infect cabbage than tomato. Especially, the Pcal KB211 strain used in this study was isolated from broccoli, a Brassicaceae species [24]. Moreover, the Pcal host range mostly comprises Brassicaceae species [4]. Therefore, Pcal has likely evolved its virulence along with Brassicaceae species more than tomato. There are several possible reasons why Pcal can multiply to great levels in cabbage. First, Pcal virulence factors are suitable for suppressing cabbage plant defenses. Although it is difficult to compare the defense strength of different plants, it might be possible that a sufficient population is required to suppress plant immunity. Second, Pcal has a great ability to grow on the nutrients inside the cabbage apoplast. Pst PT23 populations reached greater densities when grown in tomato (native host plants) than A. thaliana (alternative host plants) [25]. Third, the regulation of hrpL is disrupted in tomato. HrpL regulates several virulence-related genes, including the TTSS component, effectors, and COR biosynthesis [26,27]. Wang et al. (2020) demonstrated that secondary metabolites produced by A. thaliana directly suppress the expression of T3SS [28]. Thus, it is tempting to speculate that differences in metabolites from each plant may differentially affect hrpL regulation. To elucidate the unique infection strategies that each plant pathogenic bacteria employs to infect its native host will be a challenge for future research.
Furthermore, Pst populations in tomato inoculated at 5 × 103 CFU/mL inoculum levels were greater than with 5 × 106 CFU/mL inoculum levels at 3 dpi (Figure 3). Moreover, the bacterial virulence genes were also not induced when we inoculated with 5 × 103 CFU/mL (Figure 4). Together, when the bacterial entry is lesser (e.g. 5 × 103 CFU/mL), bacteria avoid plant defense induction and can multiply to increased levels and maintain their population without using virulence factors. Thus, these results demonstrate that the entry of lesser populations, which were not enough to induce plant resistance, might be the best way to multiply without using energy to fight with plants. Moreover, the bacterial populations in tomato inoculated at 5 × 103 CFU/mL reached the same levels as that of tomato inoculated at greater concentrations within three days (Figure 3B). Therefore, bacterial density might not be related to regulating virulence factors well in Pst virulence.
T3SS- and COR-related gene expressions were upregulated in Pcal–cabbage and Pst–tomato interactions, especially when we inoculated with 5 × 105 CFU/mL inoculum levels (Figure 2 and Figure 4). Conversely, T3SS- and COR-related gene expressions were not upregulated during Pcal infection of tomato, compared to that during Pcal infection of cabbage (Figure 2). These results are consistent with our results that Pcal multiplied to greater levels in cabbage compared to tomato (Figure 1B,D). Our results also showed that the Pcal T3SS mutant and Pst hrcC mutant populations were significantly reduced at 5 × 103 CFU/mL inoculum levels in cabbage and tomato, respectively (Figure 5). Although the bacterial populations of COR mutants showed no significant differences, the COR mutant did not multiply well inside the plants after syringe inoculation at 1 dpi [8]. The trade-off between virulence factor production and growth with nutrients might be present in bacterial infection processes [29]. Although further analysis will be needed, it is tempting to speculate that if a bacterial pathogen exhibits excess virulence, they cannot proliferate effectively. Thus, maintaining a balance is likely the most beneficial strategy for bacterial pathogens.
Moreover, Pst fliC expression was reduced in tomato at greater inoculum levels (Figure 4), while that of Pcal fliC did not change at different bacterial concentrations (Figure 2). These results indicate that Pst down-regulates fliC expression when the bacterial concentration is increased. Furthermore, Pcal fliC expression during tomato infection was lower than during cabbage infection (Figure 2). These differences might be caused by the fact that there are a variety of bacterial flagellin epitopes and their receptors. The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). Pcal has flg22 variants that can act as antagonists of plant defense activation [30,31]. Once plant pathogenic bacteria enter the plant, they down-regulate flagellin-related genes at the late infection stage, thereby reducing the production of flagellin-derived elicitors. Repression of motility-associated genes in plants was reported for P. syringae [14,32,33]. Since Pcal FliC was not recognized in host cabbage, it is tempting to speculate that regulation of fliC expression was not needed during Pcal infection of cabbage. Recently, the receptor FLS3 for flgII-28 was identified in tomato, while A. thaliana lacks FLS3. This suggests that cabbage might also lack the FLS3 for the flgII-28 receptor. Consequently, it is possible Pcal flgII-28 is specifically recognized by tomato.
Furthermore, Pcal ΔfliC did not multiply well at 5 × 103 CFU/mL inoculum levels, whereas Pst fliC multiplied similarly to WT (Figure 5). fliC deletion results in motility loss in Pcal and Pst [18,34]. As discussed above, Pcal flg22 can evade plant recognition [18,30,31]. Therefore, Pcal ΔfliC abolishes motility, leading to reduced virulence. Conversely, Pst ΔfliC may evade plant recognition despite losing motility, resulting in similar multiplication rates as the WT. Consequently, no differences in virulence were observed between Pst WT and ΔfliC. Further studies are necessary to understand the relationships between flagellin-mediated PTI and flagella-related bacterial virulence in these interactions.

5. Conclusions

When we inoculated with great concentrations (5 × 105 and 5 × 106 CFU/mL), the bacterial populations grew to increased density but could not maintain their growth at 5 dpi. This fact implies that bacterial pathogens do not want excess growth. Thus, bacterial pathogens need to regulate virulence factors properly depending on the situation. Although we still do not have an idea of what bacterial pathogens prioritize during infection, revealing the spatiotemporal regulation mechanisms of virulence factors will lead to the answer of what is best for bacterial pathogens. Furthermore, our data highlight different bacterial pathogen infection strategies against different host plants. Although further investigation is still needed, comparing the infection strategies of different pathovars and on different host plans will elucidate pathogenicity.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/bacteria3040026/s1, Figure S1: Pcal growth comparison in cabbage and in tomato; Figure S2: Pcal and Pst growth comparison in tomato; Table S1: Bacterial strains used in this study; Table S2: Primer sets used in this study.

Author Contributions

Conceptualization, Y.I. and N.S.; investigation, K.Y., G.U., Y.I. and N.S; writing—original draft preparation, N.S.; writing—review and editing, Y.I.; visualization, Y.I. and N.S; funding acquisition, Y.I. and N.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Japan Society for the Promotion of Science, grant number 19K06045 (Y.I.) and 21J10765 (N.S.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We thank Christina Baker Starrman for editing this manuscript. Pcal KB211 was kindly given from the Nagano vegetable and ornamental crops experiment station, Nagano, Japan. Pst DC3000 was kindly given from Fumiaki Katagiri at the University of Minnesota.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of this manuscript; or in the decision to publish the results.

References

  1. Xin, X.F.; Kvitko, B.; He, S.Y. Pseudomonas syringae: What it takes to be a pathogen. Nat. Rev. Microbiol. 2018, 16, 316–328. [Google Scholar] [CrossRef]
  2. Xin, X.-F.; He, S.Y. Pseudomonas syringae pv. tomato DC3000: A model pathogen for probing disease susceptibility and hormone signaling in plants. Annu. Rev. Phytopathol. 2013, 51, 473–498. [Google Scholar] [CrossRef] [PubMed]
  3. Ishiyama, Y.; Yamagishi, N.; Ogiso, H.; Fujinaga, M.; Takikawa, Y. Bacterial brown spot on Avena storigosa Schereb. caused by Pseudomonas syringae pv. alisalensis. J. Gen. Plant Pathol. 2013, 79, 155–157. [Google Scholar] [CrossRef]
  4. Takikawa, Y.; Takahashi, F. Bacterial leaf spot and blight of crucifer plants (Brassicaceae) caused by Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. J. Gen. Plant Pathol. 2014, 80, 466–474. [Google Scholar] [CrossRef]
  5. Sakata, N.; Ino, T.; Hayashi, C.; Ishiga, T.; Ishiga, Y. Controlling stomatal aperture, a potential strategy for managing plant bacterial disease. Plant Sci. 2023, 327, 111534. [Google Scholar] [CrossRef]
  6. Baltrus, D.A.; Nishimura, M.T.; Romanchuk, A.; Chang, J.H.; Mukhtar, M.S.; Cherkis, K.; Roach, J.; Grant, S.R.; Jones, C.D.; Dangl, J.L. Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates. PLoS Pathog. 2011, 7, e1002132. [Google Scholar] [CrossRef]
  7. Bender, C.L.; Alarcón-Chaidez, F.; Gross, D.C. Pseudomonas syringae phytotoxins: Mode of action, regulation, and biosynthesis by peptide and polyketide synthetases. Microbiol. Mol. Biol. Rev. 1999, 63, 266–292. [Google Scholar] [CrossRef]
  8. Sakata, N.; Ishiga, T.; Masuo, S.; Hashimoto, Y.; Ishiga, Y. Coronatine contributes to Pseudomonas cannabina pv. alisalensis virulence by overcoming both stomatal and apoplastic defenses in dicot and monocot Plants. Mol. Plant. Microbe. Interact. 2021, 34, 746–757. [Google Scholar] [CrossRef]
  9. Melotto, M.; Underwood, W.; Koczan, J.; Nomura, K.; He, S.Y. Plant stomata function in innate immunity against bacterial invasion. Cell 2006, 126, 969–980. [Google Scholar] [CrossRef]
  10. Melotto, M.; Underwood, W.; He, S.Y. Role of stomata in plant innate immunity and foliar bacterial diseases. Annu. Rev. Phytopathol. 2008, 46, 101–122. [Google Scholar] [CrossRef]
  11. Ichihara, A.; Shiraishi, K.; Sato, H.; Sakamura, S.; Nishiyama, K.; Sakai, R.; Furusaki, A.; Matsumoto, T. The structure of coronatine. J. Am. Chem. Soc. 1977, 99, 636–637. [Google Scholar] [CrossRef]
  12. Peñaloza-Vázquez, A.; Bender, C.L. Characterization of CorR, a transcriptional activator which is required for biosynthesis of the phytotoxin coronatine. J. Bacteriol. 1998, 180, 6252–6259. [Google Scholar] [CrossRef] [PubMed]
  13. Markel, E.; Stodghill, P.; Bao, Z.; Myers, C.R.; Swingle, B. AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000. J. Bacteriol. 2016, 198, 2330–2344. [Google Scholar] [CrossRef] [PubMed]
  14. Bao, Z.; Wei, H.-L.; Ma, X.; Swingle, B. Pseudomonas syringae AlgU downregulates flagellin gene expression, helping evade plant immunity. J. Bacteriol. 2020, 202, e00418-19. [Google Scholar] [CrossRef]
  15. Zhang, Y.; Fu, M.; Wang, Q.; Zhang, L.; Chang, X.; Zhang, L. Role of the sigma factor AlgU in regulating growth, virulence, motility, exopolysaccharide production, and environmental stress adaptation of Pseudomonas syringae pv. actinidiae QSY6. Phytopathol. Res. 2024, 6, 26. [Google Scholar] [CrossRef]
  16. Taguchi, F.; Suzuki, T.; Inagaki, Y.; Toyoda, K.; Shiraishi, T.; Ichinose, Y. The siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection. J. Bacteriol. 2010, 192, 117–126. [Google Scholar] [CrossRef]
  17. Swingle, B.; Thete, D.; Moll, M.; Myers, C.R.; Schneider, D.J.; Cartinhour, S. Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other Pseudomonads. Mol. Microbiol. 2008, 68, 871–889. [Google Scholar] [CrossRef]
  18. Clarke, C.R.; Chinchilla, D.; Hind, S.R.; Taguchi, F.; Miki, R.; Ichinose, Y.; Martin, G.B.; Leman, S.; Felix, G.; Vinatzer, B.A. Allelic Variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility. New Phytol. 2013, 200, 847–860. [Google Scholar] [CrossRef]
  19. Sun, W.; Dunning, F.M.; Pfund, C.; Weingarten, R. Within-species flagellin polymorphism in Xanthomonas campestris pv campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2–Dependent defense. Plant Cell 2006, 18, 764–779. [Google Scholar] [CrossRef]
  20. Sakata, N.; Ishiga, T.; Saito, H.; Nguyen, V.T.; Ishiga, Y. Transposon mutagenesis reveals Pseudomonas cannabina pv. alisalensis optimizes its virulence factors for pathogenicity on different hosts. PeerJ 2019, 2019, e7698. [Google Scholar] [CrossRef]
  21. Elizabeth, S.V.; Bender, C.L. The phytotoxin coronatine from Pseudomonas syringae pv. tomato DC3000 functions as a virulence factor and influences defence pathways in edible brassicas. Mol. Plant Pathol. 2007, 8, 83–92. [Google Scholar] [CrossRef] [PubMed]
  22. King, E.O.; Ward, M.K.; Raney, D.E. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 1954, 44, 301–307. [Google Scholar] [PubMed]
  23. Ishiga, Y.; Ichinose, Y. Pseudomonas syringae pv. tomato OxyR is required for virulence in tomato and Arabidopsis. Mol. Plant. Microbe. Interact. 2016, 29, 119–131. [Google Scholar] [CrossRef] [PubMed]
  24. Takahashi, F.; Ogiso, H.; Fujinaga, M.; Ishiyama, Y.; Inoue, Y.; Shirakawa, T.; Takikawa, Y. First report of bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis in Japan. J. Gen. Plant Pathol. 2013, 79, 260–269. [Google Scholar] [CrossRef]
  25. Meaden, S.; Koskella, B. Adaptation of the pathogen, Pseudomonas syringae, during experimental evolution on a native vs. alternative host plant. Mol. Ecol. 2017, 26, 1790–1801. [Google Scholar] [CrossRef]
  26. Fouts, D.E.; Abramovitch, R.B.; Alfano, J.R.; Baldo, A.M.; Buell, C.R.; Cartinhour, S.; Chatterjee, A.K.; D’Ascenzo, M.; Gwinn, M.L.; Lazarowitz, S.G.; et al. Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor. Proc. Natl. Acad. Sci. USA 2002, 99, 2275–2280. [Google Scholar] [CrossRef]
  27. Sreedharan, A.; Penaloza-Vazquez, A.; Kunkel, B.N.; Bender, C.L. CorR regulates multiple components of virulence in Pseudomonas syringae pv. tomato DC3000. Mol. Plant. Microbe. Interact. 2006, 19, 768–779. [Google Scholar] [CrossRef]
  28. Wang, W.; Yang, J.; Zhang, J.; Liu, Y.X.; Tian, C.; Qu, B.; Gao, C.; Xin, P.; Cheng, S.; Zhang, W.; et al. An Arabidopsis secondary metabolite directly targets expression of the bacterial type III secretion system to inhibit bacterial virulence. Cell Host Microbe 2020, 27, 601–613.e7. [Google Scholar] [CrossRef]
  29. Sakata, N.; Ishiga, T.; Ishiga, Y. Pseudmonas cannabina pv. alisalensis TrpA is required for virulence in multiple host plants. Front. Microbiol. 2021, 12, 659734. [Google Scholar] [CrossRef]
  30. Colaianni, N.R.; Parys, K.; Lee, H.-S.; Conway, J.M.; Kim, N.H.; Edelbacher, N.; Mucyn, T.S.; Madalinski, M.; Law, T.F.; Jones, C.D.; et al. A complex immune response to flagellin epitope variation in commensal communities. Cell Host Microbe 2021, 29, 635–649.e9. [Google Scholar] [CrossRef]
  31. Parys, K.; Colaianni, N.R.; Lee, H.S.; Hohmann, U.; Edelbacher, N.; Trgovcevic, A.; Blahovska, Z.; Lee, D.; Mechtler, A.; Muhari-Portik, Z.; et al. Signatures of antagonistic pleiotropy in a bacterial flagellin epitope. Cell Host Microbe 2021, 29, 620–634.e9. [Google Scholar] [CrossRef] [PubMed]
  32. Lovelace, A.H.; Smith, A.; Kvitko, B.H. Pattern-triggered immunity alters the transcriptional regulation of virulence-associated genes and induces the sulfur starvation response in Pseudomonas syringae pv. tomato DC3000. Mol. Plant. Microbe. Interact. 2018, 31, 750–765. [Google Scholar] [CrossRef] [PubMed]
  33. Nobori, T.; Velásquez, A.C.; Wu, J.; Kvitko, B.H.; Kremer, J.M.; Wang, Y.; He, S.Y.; Tsuda, K. Transcriptome landscape of a bacterial pathogen under plant immunity. Proc. Natl. Acad. Sci. USA 2018, 115, E3055–E3064. [Google Scholar] [CrossRef]
  34. Sakata, N.; Shiraishi, N.; Saito, H.; Komoto, H.; Ishiga, T.; Usuki, G.; Yamashita, Y.; Ishiga, Y. Covering cabbage leaves with cellulose nanofiber confers resistance against Pseudomonas cannabina pv. alisalensis. J. Gen. Plant Pathol. 2022, 89, 53–60. [Google Scholar] [CrossRef]
Bacteria 03 00026 g001
Figure 1. Disease phenotype and bacterial populations of Pseudomonas cannabina pv. alisalnesis (Pcal) in cabbage and tomato. Disease symptoms in cabbage (A) and in tomato (B) syringe-inoculated with Pcal (5 × 103, 5 × 104, 5 × 105, and 5 × 106 CFU/mL). Bacterial populations syringe-inoculated with Pcal (5 × 103 (C), 5 × 104 (D), 5 × 105 (E), and 5 × 106 (F) CFU/mL). Bacterial populations in the plant were evaluated at 0, 1, 2, 3, 4, and 5 dpi. The leaves were photographed 1, 2, 3, 4, and 5 dpi. Vertical bars indicate the standard error for three independent experiments. Asterisks indicate a significant difference between bacterial populations in cabbage and tomato in a t-test (* p < 0.05, ** p < 0.01). The scale bar shows 2 cm.
Figure 1. Disease phenotype and bacterial populations of Pseudomonas cannabina pv. alisalnesis (Pcal) in cabbage and tomato. Disease symptoms in cabbage (A) and in tomato (B) syringe-inoculated with Pcal (5 × 103, 5 × 104, 5 × 105, and 5 × 106 CFU/mL). Bacterial populations syringe-inoculated with Pcal (5 × 103 (C), 5 × 104 (D), 5 × 105 (E), and 5 × 106 (F) CFU/mL). Bacterial populations in the plant were evaluated at 0, 1, 2, 3, 4, and 5 dpi. The leaves were photographed 1, 2, 3, 4, and 5 dpi. Vertical bars indicate the standard error for three independent experiments. Asterisks indicate a significant difference between bacterial populations in cabbage and tomato in a t-test (* p < 0.05, ** p < 0.01). The scale bar shows 2 cm.
Bacteria 03 00026 g001
Bacteria 03 00026 g002
Figure 2. Pcal virulence-related gene expression profiles during infection in cabbage and tomato. Expression profiles of hrpL (A), hopM1 (B), avrPto (C), corR (D), cfl (E), cmaA (F), pvdS (G), algU (H), and fliC (I) genes in syringe-inoculated cabbage plants (5 × 103 and 5 × 105 CFU/mL) at 2, 3, and 4 days after inoculation. Total RNA was extracted for use in real-time quantitative reverse transcription PCR with gene-specific primer sets. Expression was normalized using oprF and recA. Vertical bars indicate the standard error for three biological replicates. Asterisks indicate a significant difference between the gene expression profiles in plants inoculated at 5 × 103 and 5 × 105 CFU/mL in a t-test (* p < 0.05, ** p < 0.01).
Figure 2. Pcal virulence-related gene expression profiles during infection in cabbage and tomato. Expression profiles of hrpL (A), hopM1 (B), avrPto (C), corR (D), cfl (E), cmaA (F), pvdS (G), algU (H), and fliC (I) genes in syringe-inoculated cabbage plants (5 × 103 and 5 × 105 CFU/mL) at 2, 3, and 4 days after inoculation. Total RNA was extracted for use in real-time quantitative reverse transcription PCR with gene-specific primer sets. Expression was normalized using oprF and recA. Vertical bars indicate the standard error for three biological replicates. Asterisks indicate a significant difference between the gene expression profiles in plants inoculated at 5 × 103 and 5 × 105 CFU/mL in a t-test (* p < 0.05, ** p < 0.01).
Bacteria 03 00026 g002
Bacteria 03 00026 g003
Figure 3. Disease phenotype and Pseudomonas syringae pv. tomato (Pst) populations in tomato. Disease symptoms (A) and bacterial populations (B) in tomato syringe-inoculated with Pst (5 × 103, 5 × 104, 5 × 105, and 5 × 106 CFU/mL). Bacterial populations in the plant were evaluated at 0, 1, 2, 3, 4, and 5 dpi. The leaves were photographed 1, 2, 3, 4, and 5 dpi. Vertical bars indicate the standard error for three independent experiments. Different letters indicate a significant difference among treatments based on a Tukey’s honestly significant difference test (p < 0.05). The scale bar shows 2 cm.
Figure 3. Disease phenotype and Pseudomonas syringae pv. tomato (Pst) populations in tomato. Disease symptoms (A) and bacterial populations (B) in tomato syringe-inoculated with Pst (5 × 103, 5 × 104, 5 × 105, and 5 × 106 CFU/mL). Bacterial populations in the plant were evaluated at 0, 1, 2, 3, 4, and 5 dpi. The leaves were photographed 1, 2, 3, 4, and 5 dpi. Vertical bars indicate the standard error for three independent experiments. Different letters indicate a significant difference among treatments based on a Tukey’s honestly significant difference test (p < 0.05). The scale bar shows 2 cm.
Bacteria 03 00026 g003
Bacteria 03 00026 g004
Figure 4. Pst virulence-related gene expression profiles during infection in cabbage and tomato. Expression profiles of hrpL (A), hopM1 (B), avrPto (C), corR (D), cfl (E), cmaB (F), pvdS (G), algU (H), and fliC (I) genes in syringe-inoculated cabbage plants (5 × 103 and 5 × 105 CFU/mL) at 2, 3, and 4 days after inoculation. Total RNA was extracted for use in real-time quantitative reverse transcription PCR with gene-specific primer sets. Expression was normalized using oprI, proC, and rpoD. Vertical bars indicate the standard error for three biological replicates. Different letters indicate a significant difference among treatments based on Tukey’s honestly significant difference test (p < 0.05).
Figure 4. Pst virulence-related gene expression profiles during infection in cabbage and tomato. Expression profiles of hrpL (A), hopM1 (B), avrPto (C), corR (D), cfl (E), cmaB (F), pvdS (G), algU (H), and fliC (I) genes in syringe-inoculated cabbage plants (5 × 103 and 5 × 105 CFU/mL) at 2, 3, and 4 days after inoculation. Total RNA was extracted for use in real-time quantitative reverse transcription PCR with gene-specific primer sets. Expression was normalized using oprI, proC, and rpoD. Vertical bars indicate the standard error for three biological replicates. Different letters indicate a significant difference among treatments based on Tukey’s honestly significant difference test (p < 0.05).
Bacteria 03 00026 g004
Bacteria 03 00026 g005
Figure 5. Disease phenotype and bacterial populations of Pcal and Pst in cabbage and tomato, respectively. Disease symptoms (A) and bacterial populations (B–D) in cabbage syringe-inoculated with Pcal WT, COR mutant (ΔcmaA), T3SS mutant (NB35), and flagellin mutant (ΔfliC). Disease symptoms (E) and bacterial populations (F–H) in tomato syringe-inoculated with Pst WT, COR mutant (DB29), T3SS mutant (hrcC mutant), and flagellin mutant (ΔfliC). Cabbage and tomato were syringe-inoculated with Pcal and Pst (5 × 103 (B and F), 5 × 105 (C and G), and 5 × 107 (D and H) CFU/mL). Bacterial populations in the plant were evaluated at 0 and 3 dpi. The leaves were photographed at 3 dpi. Vertical bars indicate the standard error for three independent experiments. Different letters indicate a significant difference among treatments based on a Tukey’s honestly significant difference test (p < 0.05). The scale bar shows 2 cm.
Figure 5. Disease phenotype and bacterial populations of Pcal and Pst in cabbage and tomato, respectively. Disease symptoms (A) and bacterial populations (B–D) in cabbage syringe-inoculated with Pcal WT, COR mutant (ΔcmaA), T3SS mutant (NB35), and flagellin mutant (ΔfliC). Disease symptoms (E) and bacterial populations (F–H) in tomato syringe-inoculated with Pst WT, COR mutant (DB29), T3SS mutant (hrcC mutant), and flagellin mutant (ΔfliC). Cabbage and tomato were syringe-inoculated with Pcal and Pst (5 × 103 (B and F), 5 × 105 (C and G), and 5 × 107 (D and H) CFU/mL). Bacterial populations in the plant were evaluated at 0 and 3 dpi. The leaves were photographed at 3 dpi. Vertical bars indicate the standard error for three independent experiments. Different letters indicate a significant difference among treatments based on a Tukey’s honestly significant difference test (p < 0.05). The scale bar shows 2 cm.
Bacteria 03 00026 g005
Table 1. Growth rate (µ) from 0 to 2 days.
Table 1. Growth rate (µ) from 0 to 2 days.
5 × 1035 × 1045 × 1055 × 106
Cabbage0.0820.0800.0740.068
Tomato0.0680.0630.0430.034
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Sakata, N.; Usuki, G.; Yamamoto, K.; Ishiga, Y. Comparative Analysis of Infection Strategies of Pseudomonas cannabina pv. alisalensis and P. syringae pv. tomato in Different Host Plants. Bacteria 2024, 3, 379-389. https://doi.org/10.3390/bacteria3040026

AMA Style

Sakata N, Usuki G, Yamamoto K, Ishiga Y. Comparative Analysis of Infection Strategies of Pseudomonas cannabina pv. alisalensis and P. syringae pv. tomato in Different Host Plants. Bacteria. 2024; 3(4):379-389. https://doi.org/10.3390/bacteria3040026

Chicago/Turabian Style

Sakata, Nanami, Giyu Usuki, Kanon Yamamoto, and Yasuhiro Ishiga. 2024. "Comparative Analysis of Infection Strategies of Pseudomonas cannabina pv. alisalensis and P. syringae pv. tomato in Different Host Plants" Bacteria 3, no. 4: 379-389. https://doi.org/10.3390/bacteria3040026

APA Style

Sakata, N., Usuki, G., Yamamoto, K., & Ishiga, Y. (2024). Comparative Analysis of Infection Strategies of Pseudomonas cannabina pv. alisalensis and P. syringae pv. tomato in Different Host Plants. Bacteria, 3(4), 379-389. https://doi.org/10.3390/bacteria3040026

Article Metrics

Back to TopTop