Abstract
Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is a serious threat to kiwifruit production. Highly virulent strains of Psa biovar3 (Psa3) have spread rapidly to kiwifruit production areas worldwide. Therefore, there is an urgent need to develop critical management strategies for bacterial canker based on dissecting the interactions between Psa and kiwifruit. Here, we developed a rapid and reliable flood-inoculation method using kiwifruit seedlings grown on Murashige and Skoog medium. This method has several advantages over inoculation of conventional soil-grown plants. We demonstrated the utility of a kiwifruit seedling assay to study the virulence of Psa biovars and Psa3 virulence factors, including the type III secretion system (T3SS). Kiwifruit seedlings inoculated with Psa3 developed severe necrosis within 1 week, whereas those inoculated with a T3SS-deficient hrcN mutant of Psa3 did not. This method was also useful for analyzing expression profiles of genes involved in Psa3 virulence during infection, and revealed that the expression of genes encoding the T3SS and type III secreted effectors were strongly induced in planta. Our results indicate that the T3SS has an important role in Psa3 virulence, and the flood-inoculation assay using kiwifruit seedling is suitable for analyzing Psa and kiwifruit interactions.
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Introduction
Pseudomonas syringae induces a variety of symptoms such as leaf spots, cankers, galls, wilt, and blights on different plants, and can be classified into more than 50 pathovars (pv.) based on their host plant specificities and disease symptoms (Mansfield et al. 2012; Xin and He 2013). P. syringae pv. actinidiae (Psa), causal agent of bacterial canker of kiwifruit, is an economically important pathogen worldwide. First reported in Japan, Psa causes severe damage to kiwifruit plants and decreases yields under optimal conditions (Scortichini et al. 2012; Takikawa et al. 1989). Psa is organized into five biovars including biovar 1, 2, 3, 5, and 6 based on biochemical, pathogenicity, and molecular characteristics of the Psa strains (Fujikawa and Sawada 2016, 2019). The emergence of Psa3 strains with enhanced virulence was reported in 2008 in Italy, and a pandemic spread rapidly to kiwifruit production areas worldwide (Scortichini et al. 2012). Major control strategies include chemical treatments such as copper fungicides and antibiotics (Cameron and Sarojini 2013), but they are associated with potential risks such as induction of Psa resistance strains, phytotoxicity, and chemical residues in fruits (Nakajima et al. 2002; Serizawa et al. 1989; Vanneste and Voyle 2003). Breeding for resistance is another control strategy, but cultivar development is slow (Kisaki et al. 2019). Therefore, alternative management strategies based on the dissection of the dynamic interactions between Psa and kiwifruit are urgently needed.
Plants have been inoculated with P. syringae using syringe pressure infiltration, vacuum infiltration, and sprays and dips to investigate the molecular basis of plant–pathogen interactions (Katagiri et al. 2002). Syringe pressure infiltration is the most commonly used method (Kisaki et al. 2019). Conventional soil-grown plant inoculation assays have included stem puncture inoculation, syringe pressure infiltration, and spray inoculation were used to study the interactions between Psa and kiwifruit (Bartoli et al. 2015; Gao et al. 2016; Kisaki et al. 2019). However, to functionally investigate the molecular interactions between plants and P. syringae, the inoculation assay should mimic natural infection and be suitable for high-throughput assays. P. syringae has two lifestyles, an epiphytic phase on the plant leaf surface and an endophytic phase in the apoplastic space (Melotto et al. 2008; Xin and He 2013). The bacterium generally enters host tissues through natural openings such as stomata and multiplies in the apoplast to cause disease in nature (Lindow and Brandl 2003).
In previous studies, we developed a flood-inoculation method to investigate the interactions of plants with P. syringae pv. tomato DC3000 (Ishiga et al. 2011; Uppalapati et al. 2008). To establish a robust assay to study Psa and kiwifruit interactions, in the present study, we developed a rapid and reliable flood-inoculation method using kiwifruit seedlings that had been grown in Murashige and Skoog (MS) medium for 4 weeks. We also demonstrated that this method is suitable to study Psa virulence mechanisms.
Materials and methods
Plant materials and growth conditions
Kiwifruit (Actinidiae delisiosa) cv. Hayward plants were used for all experiments. Kiwifruit seeds (100–200) were surface-sterilized in 70% (v/v) ethanol for 5 min in a Falcon tube (50 ml), then in 5% (v/v) sodium hypochlorite (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 0.1% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Seeds were then washed with sterile distilled H2O at least four times and incubated in water at 4 °C overnight. Surface sterilization and washing were repeated, and seeds were germinated on one-half strength MS medium (FUJIFILM Wako Pure Chemical Corporation) containing 1% (w/v) sucrose and Gamborg vitamins (Sigma-Aldrich) solidified with 0.3% (w/v) Phytagel (Sigma-Aldrich) in deep Petri plates (100 mm × 25 mm). Four-week-old kiwifruit seedlings were grown at 24 °C with a light intensity of 150–200 μmol/(m2 sec) and 12 h light/12 h dark before use. Six-week-old kiwifruit plants were grown in Berger BM2 Germinating Mix soil (Berger, Saint-Modeste, QC, Canada) at 24 °C with a light intensity of 200 μmol/(m2 sec) and 16 h light/8 h dark to use for spray inoculation.
Bacterial strains and growth conditions
All bacterial strains and plasmids are shown in Table 1. Pseudomonas syringae pv. actinidiae biovar 1 (Psa1; MAFF 613022), biovar 3 (Psa3; MAFF 212115), biovar 5 (Psa5; MAFF 212056), and biovar 6 (Psa6; MAFF 212133) were a gift from NARO Genebank, Ibaraki, Japan. Psa biovars were used as the pathogenic strains to inoculate kiwifruit plants. The Psa3 hrcN mutant defective in the type III secretion system (T3SS) was generated by conjugation with Escherichia coli S17-1, which possesses pBSLC1 (Sawada et al. 2018; Schäfer et al. 1994), and was used as a reduced-virulence mutant of Psa3. The pDSK-GFPuv vector was introduced into each Psa biovar by electroporation (Wang et al. 2007). All Psa strains were grown at 28 °C on King’s B (KB) (King et al. 1954) agar. For inoculation, Luria–Bertani (LB) (Sambrook et al. 1989) broth was used to grow bacterial cultures from plates for 18 h at 28 °C. Before inoculation, bacteria were suspended in sterile distilled H2O, and the bacterial cell densities at 600 nm (OD600) were measured using a JASCO V-730 spectrophotometer (JASCO, Tokyo, Japan).
Bacterial inoculation
A flood-inoculation method that we developed previously (Ishiga et al. 2017) to infect Arabidopsis seedlings with P. syringae pathogens was modified to develop a kiwifruit seedling inoculation method conducive to observing disease symptoms. Briefly, 40 ml of bacterial suspension (OD600 of 0.2) in sterile distilled H2O containing 0.025% (v/v) Silwet L-77 (OSI Specialties Inc., Danbury, CT, USA) was dispensed onto a plate containing 4-week-old kiwifruit seedlings, and the plates were incubated for 2–3 min at room temperature. After the bacterial suspension was removed by decantation, plates containing inoculated plants were sealed with 3 M Micropore 2.5 cm surgical tape (3 M, St. Paul, MN, USA) and incubated at 22 °C with a light intensity of 150–200 μmol/(m2 sec) and 12 h light/12 h dark. Symptoms were observed at 1- and 2-weeks post-inoculation (wpi). In each experiment, more than six plants were evaluated, and each experiment was repeated at least three times.
For spray inoculation, 6-week-old kiwifruit plants were sprayed to runoff with a bacterial suspension (OD600 of 0.2) in sterile distilled water containing 0.025% Silwet L-77. The plants were then incubated in growth chambers at approximately 100% RH for the first 24 h, then at approximately 70% RH for the rest of the experiment. The inoculated plants were observed for 1 wpi for symptom development.
To determine the bacterial growth in kiwifruit seedlings or leaves, we measured the internal bacterial population at 2 wpi. Inoculated seedlings or leaves were collected, and the inoculated plants were weighed. Seedlings or leaves were then surface-sterilized with 10% H2O2 for 3 min, then washed three times with sterile distilled water. Plants were then homogenized in sterile distilled water, and the diluted samples were plated onto solid KB agar. Bacterial colony forming units (CFU) were normalized as CFU/mg using the total mass of the inoculated plants. The bacterial population at 0 day was estimated using leaves harvested 1 h post-inoculation (hpi). The bacterial populations were evaluated in three independent experiments.
Real-time quantitative RT-PCR
For expression profiles of Psa3 genes in culture or during infection, we incubated Psa3 in LB broth for 3 h or flood-inoculated kiwifruit plants for 24 and 48 h. For expression profiles in culture conditions, Psa3 was grown in LB broth for 24 h, then adjusted to OD600 of 0.1 with fresh LB broth and grown for 3 h to investigate the expression profiles during the exponential phase, not the stationary phase. Total RNA was extracted from kiwifruit seedlings by an ultrasonication method as described (Yu et al. 2013). Briefly, 12 plants were collected, immediately submerged in 5 ml of RNAlater Stabilization Solution (Thermo Fisher Scientific, Waltham, MA, USA), sonicated for 7 min, then plants were removed from the solution. The bacterial cells in the suspension were harvested by centrifugation at 12,000 rpm for 2 min, and cell pellets were used for subsequent purification. Total RNA was extracted using Reliaprep (Promega, Madison, WI, USA) and the manufacturer’s protocol. Two micrograms of total RNA was treated with gDNA Remover (TOYOBO, Osaka, Japan) to eliminate genomic DNA, and the DNase-treated RNA was reverse transcribed using the ReverTra Ace qPCR RT Master Mix (TOYOBO). The cDNA (1:20) was then used for qRT-PCR that was performed using the primers shown in supplementary Table S1 with THUNDERBIRD® SYBR qPCR Mix (TOYOBO) on a Thermal Cycler Dice Real Time System (TaKaRa, Kusatsu, Japan). Three genes encoding fructose-bisphosphate aldolase, chromosome participating protein ParA, and a TetR family transcriptional regulator were used for normalizing the results (McAtee et al. 2018).
Results
Flood inoculation of seedlings to study Psa–kiwifruit interactions
For standardizing the seedling flood-inoculation assay and testing whether Psa multiplies and causes disease symptoms similar to adult plants grown in soil, 4-week-old kiwifruit seedlings (containing two to four leaves except for the cotyledon) were grown on MS plates. Around 80–100 seeds were planted on each MS plate (Fig. 1a), which germinated 2 weeks after planting when exposed to light (Fig. 1b). Six small seedlings were transferred to a new plate (Fig. 1c), and after 2 weeks of continuous growth, kiwifruit seedlings were flood-inoculated with a bacterial suspension (OD600 of 0.2) of Psa (Fig. 1d–g). We observed severe disease symptoms within 3 days, and severe necrosis by 1–2 wpi with Psa3 (Fig. 1h).
To further assess the utility of the seedling flood-inoculation assay, we next inoculated kiwifruit seedlings with Psa biovars including Psa1, Psa3, Psa5, and Psa6. Psa1- and Psa3-inoculated seedlings showed necrosis within 1 wpi, and severe disease symptoms at 2 wpi (Fig. 2a). Among the Psa biovars, Psa3 caused the most severe symptoms (Fig. 2a). Symptoms on Psa5-inoculated seedlings developed slower during the first compared to those on Psa1- and Psa3-inoculated seedlings, but developed typical symptoms such as leaf spots at 2 wpi (Fig. 2a). Typical disease symptoms such as leaf spots can be seen in magnified images of Psa1-, Psa3-, and Psa5-inoculated seedlings at 2 wpi (Fig. 2a, bottom row). On the other hand, symptoms on seedlings-inoculated with Psa6 were only observed at 2 wpi (Fig. 2a). Mild symptoms such as chlorosis without leaf spots can be seen in the magnified image of Psa6-inoculated seedlings. These results suggest that Psa6 shows the least virulence among the Psa biovars (Fig. 2a, bottom row). Consistent with symptom development caused by the different biovars, Psa3 populations were the highest compared to those of Psa1, Psa5, and Psa6 (Fig. 2b), and Psa6 populations were the lowest. We also observed bacterial colonies in kiwifruit seedlings by labeling the Psa biovars with the GFPuv protein. Interestingly, the bacterial colonies were clearly observed in Psa1- and Psa3-inoculated seedlings at 6 days post inoculation (Fig. 2c). To evaluate the utility of the kiwifruit seedling-flood inoculation assay for studying nonhost interactions, we inoculated kiwifruit seedlings with P. syringae pv. tomato strain DC3000 (Pst DC3000). Pst DC3000 did not induce HR cell death in the healthy-looking inoculated seedlings by 1 wpi (Supplementary Fig. S1a). Pst DC3000 bacterial populations were also lower than those of Psa3 (Supplementary Fig. S1b). Together, these results suggest that the kiwifruit seedling flood-inoculation assay is a reliable method to study Psa disease progression and to evaluate in planta bacterial growth.
Kiwifruit seedling flood-inoculation assay to study a Psa virulence mutant
To evaluate the utility of the seedling flood-inoculation assay for the study of Psa3 virulence factors, we flood- or spray-inoculated kiwifruit seedlings or plants with wild-type Psa3 and a Type III secretion system (T3SS) deficient hrcN mutant. Wild-type Psa3 caused severe symptoms not only on seedlings but also on mature leaves (Fig. 3a and b). However, severe necrosis was not observed on hrcN mutant-inoculated seedlings and leaves (Fig. 3a and b). Consistent with disease development, the bacterial populations of the hrcN mutant were approximately 1000-fold lower compared to wild-type Psa3 after both inoculation methods (Fig. 3c and d). These results indicate that the T3SS has important roles in bacterial multiplication, persistence, and disease symptom development of Psa3 in kiwifruit seedlings, and in soil-grown kiwifruit plants. The kiwifruit seedling flood-inoculation assay is suitable for analyzing Psa virulence mutants.
Flood-inoculation assay of kiwifruit seedlings to study expression profiles of virulence genes during Psa infection
Pseudomonas syringae virulence genes are expressed during infection (McAtee et al. 2018; Nobori et al. 2018). We thus investigated the expression profiles of these genes in wild-type Psa3 using RT-qPCR and gene-specific primer sets according to a previous study (McAtee et al. 2018; Supplementary Table S1). We carried out expression analysis using RNAs from flood-inoculated seedlings (Fig. 4a–h) and spray-inoculated plant leaves similar to the method used for Fig. 3 (Fig. 4i–p). Our results demonstrated that expression of the alternative sigma factor hrpL, which controls the expression of T3SS effectors (T3SEs) (Fouts et al. 2002), was clearly induced in planta (in flood- and in spray-inoculated kiwifruit plants) compared to culture in LB broth (Fig. 4a and i). In addition, the expression of Psa-conserved T3SS and T3SEs genes, including hrpA1, avrRpm1, hopR1, and hopZ3 were also induced in planta (Fig. 4j–l), indicating that the T3SS is activated during Psa3 infection. We also investigated other P. syringae virulence genes (Ichinose et al. 2013; Ishiga et al. 2018; Ishiga and Ichinose 2016). The oxyR and fliC transcripts, encoding transcription factors related to oxidative stress (Ishiga and Ichinose 2016) and flagellin (Shimizu et al. 2003), respectively, were reduced in planta compared to levels produced in broth culture (Fig. 4f, g, and o). The expression of algD encoding an alginate biosynthesis enzyme (Yu et al. 1999) was induced in planta (Fig. 4h and p). Overall, we found that expression trends for most genes were the same between the two types of inoculation methods (Fig. 4). Together, our results suggest that the kiwifruit seedling flood-inoculation assay is suitable for analyzing Psa expression profiles during infection.
Discussion
Our assessments of the newly developed seedling flood-inoculation assay for kiwifruit (Fig. 1) showed that it is suitable for analyzing the virulence mechanism of Psa biovars (Fig. 2). Our method has several advantages over previous conventional soil-grown plant inoculation assays (Bartoli et al. 2015; Gao et al. 2016; Kisaki et al. 2019), including a shorter growth and incubation period, ease of inoculation and handling, uniformity of infection and disease development, and need for less growth chamber space (Fig. 1). This new method is suitable not only for dissecting the dynamic interactions between Psa and kiwifruit, but also for high-throughput screening for Psa virulence mutants, to further develop critical management strategies for bacterial canker of kiwifruit.
We observed Psa colonies in kiwifruit seedlings by labeling with a GFPuv protein. The bacterial colonies were clearly observed in Psa1- and Psa3-inoculated seedlings compared to Psa5- and Psa6-inoculated leaves (Fig. 2c). However, the population size of Psa5 was almost the same as that of Psa1 (Fig. 2b). One reason to explain the differences in the GFPuv intensity and the population size between Psa5 and Psa1 could be differences in colonization sites. P. syringae pathovars are known to have different lifestyles (Lindow and Brandl 2003; Xin and He 2013). The Psa observation system with GFPuv, which we developed in the present study, will help us spatiotemporally analyze colonization by Psa biovars and understand the Psa infection processes.
The T3SS is a key virulence component of P. syringae, because hrp/hrc mutants that block the T3SS completely eliminate virulence against susceptible host plants (Lindeberg et al. 2012; Xin and He 2013). We demonstrated that a T3SS deficient Psa hrpN mutant impaired virulence not only in kiwifruit seedlings, but also in soil-grown plants (Fig. 3), indicating that the T3SS has an important role in Psa3 virulence. In addition, several studies reported that each Psa strain has T3SEs that are conserved among biovars as well as a unique set (Fujikawa and Sawada 2016, 2019; McCann et al. 2013; Zhao et al. 2019). However, the molecular function of the T3SEs in Psa is largely unknown. Therefore, it is important to investigate the molecular basis of the T3SEs in the virulence of Psa biovars, and the flood-inoculation assay will be a robust method.
We also demonstrated that the expression of Psa3 genes encoding the T3SS and T3SEs was induced in planta compared to culture conditions in both flood- and spray-inoculated plants (Fig. 4i–l). Several transcriptome studies have been performed using different P. syringae pathovars including P. syringae pv. syringae (Pss) B728a, P. syringae pv. tomato (Pst) DC3000, and Psa3 (McAtee et al. 2018; Nobori et al. 2018; Yu et al. 2013). Yu et al. (2013) showed that expression of genes encoding the T3SS and T3SEs in Pss B728a was not strongly induced in the apoplast. In contrast, these genes were greatly upregulated in planta in comprehensive analyses of the expression profiles of Pst DC3000 and Psa3 (McAtee et al. 2018; Nobori et al. 2018). One reason to explain the differences in the T3SS and T3SEs expression profiles in planta among P. syringae pathovars is their different lifestyles. Pss is known as a particularly successful epiphyte, whereas Pst DC3000 is a weak epiphyte (Lindow and Brandl 2003; Xin and He 2013). A putative epiphytic phase was suggested for Psa3 on asymptomatic flowers and leaves (Gallelli et al. 2011; Stefani and Giovanardi 2011); however, how Psa modulates the epiphytic and endophytic phases and which genes arerequired for these phases has not been fully ascertained. Understanding Psa lifestyles will be critical to developing new management strategies for bacterial canker of kiwifruit. We believe that the flood-inoculation method will enable us to identify Psa genes required not only for virulence but also for epiphytic fitness and will provide critical clues to develop effective control strategies.
References
Bartoli C, Lamichhane JR, Berge O, Guilbaud C, Varvaro L, Balestra GM, Vinatzer BA, Morris CE (2015) A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker. Mol Plant Pathol 16:137–149
Buell CR, Joardar V, Lindeberg M, Selengut J, Paulsen IT, Gwinn ML, Dodson RJ, Deboy RT, Durkin AS, Kolonay JF, Madupu R, Daugherty S, Brinkac L, Beanan MJ, Haft DH, Nelson WC, Davidsen T, Zafar N, Zhou L, Liu J, Yuan Q, Khouri H, Fedorova N, Tran B, Russell D, Berry K, Utterback T, Van Aken SE, Feldblyum TV, D'Ascenzo M, Deng WL, Ramos AR, Alfano JR, Cartinhour S, Chatterjee AK, Delaney TP, Lazarowitz SG, Martin GB, Schneider DJ, Tang X, Bender CL, White O, Fraser CM, Collmer A (2003) The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA 100:10181–10186
Cameron A, Sarojini V (2013) Pseudomonas syringae pv. actinidiae: chemical control, resistance mechanisms and possible alternatives. Plant Pathol 63:1–11
Fouts DE, Abramovitch RB, Alfano JR, Baldo AM, Buell CR, Cartinhour S, Chatterjee AK, D’Ascenzo M, Gwinn ML, Lazarowitz SG, Lin NC, Martin GB, Rehm AH, Schneider DJ, van Dijk K, Tang X, Collmer A (2002) Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor. Proc Natl Acad Sci USA 99:2275–2280
Fujikawa T, Sawada H (2016) Genome analysis of the kiwifruit canker pathogen Pseudomonas syringae pv. actinidiae biovar 5. Sci Rep 6:21399
Fujikawa T, Sawada H (2019) Genome analysis of Pseudomonas syringae pv. actinidiae biovar 6, which produces the phytotoxins, phaseolotoxin and coronatine. Sci Rep 9:3836
Gallelli A, Talocci S, L’Aurora A, Loreti S (2011) Detection of Pseudomonas syringae pv. actinidiae, causal agent of bacterial canker of kiwifruit, from symptomless fruits and twigs, and from pollen. Phytopathol Mediterr 50:462–472
Gao X, Huang Q, Zhao Z, Han Q, Ke X, Qin H, Huang L (2016) Studies on the infection, colonization, and movement of Pseudomonas syringae pv. actinidiae in kiwifruit tissues using a GFPuv-labeled strain. PLoS One 11:e0151169
Ichinose Y, Taguchi F, Mukaihara T (2013) Pathogenicity and virulence factors of Pseudomonas syringae. J Gen Plant Pathol 79:285–296
Ishiga Y, Ichinose Y (2016) Pseudomonas syringae pv. tomato OxyR is required for virulence in tomato and Arabidopsis. Mol Plant Microbe Interact 29:119–131
Ishiga Y, Ishiga T, Uppalapati SR, Mysore KS (2011) Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant–bacterial interactions. Plant Methods 7:32
Ishiga Y, Ishiga T, Ichinose Y, Mysore KS (2017) Pseudomonas syringae flood-inoculation method in Arabidopsis. Bio-protocol 7:e2106
Ishiga T, Ishiga Y, Betsuyaku S, Nomura N (2018) AlgU contributes to the virulence of Pseudomonas syringae pv. tomato DC3000 by regulating phytotoxin coronatine production. J Gen Plant Pathol 84:189–201
Katagiri F, Thilmony R, He SY (2002) The Arabidopsis thaliana–Pseudomonas syringae interaction. Arabidopsis Book 1:e0039
King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307
Kisaki G, Tanaka S, Ishihara A, Igarashi C, Morimoto T, Hamano K, Endo A, Sugita-Konishi S, Tabuchi M, Gomi K, Ichimura K, Suezawa K, Otani M, Fukuda T, Manabe T, Fujimura T, Kataoka I, Akimitsu K (2019) Evaluation of various cultivars of Actinidia species and breeding source Actinidia rufa for resistance to Pseudomonas syringae pv. actinidiae biovar 3. J Gen Plant Pathol 84:399–406
Lindeberg M, Cunnac S, Collmer A (2012) Pseudomonas syringae type III effector repertoires: last words in endless arguments. Trends Microbiol 20:199–208
Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883
Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629
McAtee PA, Brian L, Curran B, van der Linden O, Nieuwenhuizen NJ, Chen X, Henry-Kirk RA, Stroud EA, Nardozza S, Jayaraman J, Rikkerink EHA, Print CG, Allan AC, Templeton MD (2018) Re-programming of Pseudomonas syringae pv. actinidiae gene expression during early stages of infection of kiwifruit. BMC Genomics 19:822
McCann HC, Rikkerink EH, Bertels F, Fiers M, Lu A, Rees-George J, Andersen MT, Gleave AP, Haubold B, Wohlers MW, Guttman DS, Wang PW, Straub C, Vanneste JL, Vanneste J, Rainey PB, Templeton MD (2013) Genomic analysis of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease. PLoS Pathog 9:e1003503
Melotto M, Underwood W, He SY (2008) Role of stomata in plant innate immunity and foliar bacterial diseases. Annu Rev Phytopathol 46:101–122
Nakajima M, Goto M, Hibi T (2002) Similarity between copper resistance genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato. J Gen Plant Pathol 68:68–74
Nobori T, Velásquez AC, Wu J, Kvitko BH, Kremer JM, Wang Y, He SY, Tsuda K (2018) Transcriptome landscape of a bacterial pathogen under plant immunity. Proc Natl Acad Sci USA 115:E3055–E3064
Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Harbor
Sawada T, Eguchi M, Asaki S, Kashiwagi R, Shimomura K, Taguchi F, Matsui H, Yamamoto M, Noutoshi Y, Toyoda K, Ichinose Y (2018) MexEF-OprN multidrug efflux pump transporter negatively controls N-acyl-homoserine lactone accumulation in Pseudomonas syringae pv. tabaci 6605. Mol Genet Genomics 293:907–917
Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73
Scortichini M, Marcelletti S, Ferrante P, Petriccione M, Firrao G (2012) Pseudomonas syringae pv. actinidiae: a re-emerging, multi-faceted, pandemic pathogen. Mol Plant Pathol 13:631–640
Serizawa S, Takikawa Y, Ichikawa T, Tsuyumu S, Goto M (1989) Occurrence of bacterial canker of kiwifruit in Japan: description of symptoms, isolation of the pathogen and screening of bactericides. Jpn J Phytopathol 55:427–436
Shimizu R, Taguchi F, Marutani M, Mukaihara T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) The ΔfliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 269:21–30
Stefani E, Giovanardi D (2011) Dissemination of Pseudomonas syringae pv. actinidiae through pollen and its epiphytic life on leaves and fruits. Phytopathol Mediterr 50:489–496
Takikawa Y, Serizawa S, Ichikawa T, Tsuyumu S, Goto M (1989) Pseudomonas syringae pv. actinidiae pv. nov.: the causal bacterium of canker of kiwifruit in Japan. Jpn J Phytopathol 55:437–444
Uppalapati SR, Ishiga Y, Wangdi T, Urbanczyk-Wochniak E, Ishiga T, Mysore KS, Bender CL (2008) Pathogenicity of Pseudomonas syringae pv. tomato on tomato seedlings: phenotypic and gene expression analyses of the virulence function of coronatine. Mol Plant Microbe Interact 21:383–395
Vanneste J, Voyle M (2003) Genetic basis of copper resistance in New Zealand strains of Pseudomonas syringae. New Zeal Plant Prot 56:109–112
Wang K, Kang L, Anand A, Lazarovits G, Mysore KS (2007) Monitoring in planta bacterial infection at both cellular and whole-plant levels using the green fluorescent protein variant GFPuv. New Phytol 174:212–223
Xin XF, He SY (2013) Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51:473–498
Yu J, Penaloza-Vazquez A, Chakrabarty AM, Bender CL (1999) Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae. Mol Microbiol 33:712–720
Yu X, Lund SP, Scott RA, Greenwald JW, Records AH, Nettleton D, Lindow SE, Gross DC, Beattie GA (2013) Transcriptional responses of Pseudomonas syringae to growth in epiphytic versus apoplastic leaf sites. Proc Natl Acad Sci USA 110:E425–434
Zhao Z, Chen J, Gao X, Zhang D, Zhang J, Wen J, Qin H, Guo M, Huang L (2019) Comparative genomics reveal pathogenicity-related loci in Pseudomonas syringae pv. actinidiae biovar 3. Mol Plant Pathol 20:923–942
Acknowledgements
We thank Dr. Christina Baker for editing the manuscript. Psa biovars were the gift of the NARO Genebank, Ibaraki, Japan. This work was supported in part by the JST ERATO NOMURA Microbial Community Control Project, JST, Japan.
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Figure S1
Seedling flood-inoculation assay to analyze nonhost interactions. a Disease phenotype of kiwifruit seedlings 1 week after flood inoculation with suspensions (OD600 of 0.2) of Psa3 and P. syringae pv. tomato DC3000 (Pst DC3000) containing 0.025% Silwet L-77. Scale bars, 1 cm. b Populations of Psa3 and Pst DC3000 at 0 and 1 week after flood inoculation of kiwifruit seedlings. Vertical bars indicate the standard errors for three independent experiments. Asterisks indicate a significant difference from the wild type in a t test (*P < 0.01) (PPTX 970 kb)
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Ishiga, T., Sakata, N., Nguyen, V.T. et al. Flood inoculation of seedlings on culture medium to study interactions between Pseudomonas syringae pv. actinidiae and kiwifruit. J Gen Plant Pathol 86, 257–265 (2020). https://doi.org/10.1007/s10327-020-00916-4
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DOI: https://doi.org/10.1007/s10327-020-00916-4