O ensaio de inundação de mudas facilita a rápida triagem das adesões de tomate selvagem para a resistência à bactéria Pseudomonas seringae. Este ensaio, usado em conjunto com o ensaio de crescimento bacteriano de mudas, pode auxiliar na caracterização da resistência subjacente à bactéria, e pode ser usado para mapear populações para determinar a base genética da resistência.
Artigo de método
Identificação de alta duração da resistência a Pseudomonas syringae pv. Tomate no Tomate usando Ensaio de Inundação de Mudas
Neste artigo
Aviso de errata
Important: There has been an erratum issued for this article. View Erratum Notice
Resumo
Resumo
O tomate é uma cultura agronômica importante que pode ser infectada pela Pseudomonas singae, uma bactéria Gram-negativa, resultando em doenças bacterianas. Otomate-P. sinringae pv. o patosistema de tomate é amplamente utilizado para dissecar a base genética das respostas inatas da planta e da resistência à doença. Embora a doença tenha sido gerida com sucesso por muitas décadas através da introdução do aglomerado genético Pto/PRF de Solanum pimpinellifolium em tomate cultivado, as cepas de raça 1 de P. sinringae evoluíram para superar a resistência conferida pelo grupo genético Pto/PRF e ocorrem em todo o mundo.
As espécies de tomate silvestre são importantes reservatórios de diversidade natural no reconhecimento de patógenos, pois evoluíram em diversos ambientes com diferentes pressões patogênicas. Em telas típicas para resistência a doenças no tomate selvagem, plantas adultas são usadas, o que pode limitar o número de plantas que podem ser rastreadas devido ao seu tempo de crescimento prolongado e maiores exigências de espaço de crescimento. Desenvolvemos um método para testar mudas de tomate de 10 dias de idade para resistência, o que minimiza o tempo de crescimento das plantas e o espaço da câmara de crescimento, permite uma rápida rotatividade de plantas e permite que grandes tamanhos de amostra sejam testados. Os desfechos de sobrevivência ou morte das mudas podem ser tratados como fenótipos discretos ou em uma escala de resistência definida pela quantidade de novo crescimento nas mudas sobreviventes após inundações. Este método foi otimizado para tela de mudas de tomate de 10 dias de idade para resistência a duas cepas p. sinringae e pode ser facilmente adaptado a outras cepas p. syringae.
Introdução
Pseudomonas sinringae é uma bactéria patogênica gram-negativa que infecta uma ampla gama de hospedeiros vegetais. As bactérias entram na planta hospedeira através dos estomatos ou feridas físicas e proliferam no apoplast1. As plantas desenvolveram uma resposta imune de duas camadas para proteger contra infecções por patógenos bacterianos. O primeiro nível ocorre na superfície celular da planta, onde receptores de reconhecimento de padrões na membrana celular vegetal percebem padrões moleculares altamente conservados associados ao patógeno (PAMPs) em um processo chamado imunidade desencadeada por PAMP (PTI)2. Dur....
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Protocolo
1. Preparação e uso de armário de biossegurança
- Limpe o armário de biossegurança com 70% de etanol.
- Feche a faixa e ligue a luz ultravioleta no armário de biossegurança por 15 min.
- Depois de 15 min, desligue a luz ultravioleta no armário de biossegurança. Levante a faixa e ligue o soprador por 15 min.
- Limpe todos os itens a serem usados no gabinete de biossegurança com 70% de etanol antes de colocar os itens no armário esterilizado.
- Luvas limpas ou mãos nuas com 70% de etanol antes de trabalhar no armário de biossegurança.
- Trabalhe no centro do armário de biossegurança, longe do soprador.
- Use garraf....
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Resultados
Detecção de imunidade mediada por PTORem cultivares e linhas isogênicas usando o ensaio de resistência a mudas
A Figura 5 mostra resultados representativos para as cultivares Moneymaker-PtoR e Moneymaker-PtoS 7-10 dias após a inundação com o PstDC3000. Antes da infecção, as mudas de 10 dias de idade exibiam cotyledons totalmente emergidos e expandidos e emergentes primeiras folhas verdadeiras. As mudas foram alagadas com 10 mM .......
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Discussão
Um protocolo para inoculação de inundação com PstDC3000 ou PstT1 otimizado para detectar resistência a essas cepas bacterianas em mudas de tomate é descrito. Existem vários parâmetros críticos para os resultados ótimos no ensaio de resistência às mudas, incluindo concentração bacteriana e concentração surfactante, que foram empiricamentedeterminados 22. Para o PstDC3000, a densidade óptica foi otimizada para alcançar a sobrevida completa em uma cultivar.......
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Divulgações
Os autores não têm nada para revelar.
Agradecimentos
Agradecemos a Jamie Calma por testar o efeito do volume de mídia sobre os desfechos de doenças ou resistência. Agradecemos ao Dr. Maël Baudin e ao Dr. Karl J. Scheiber do Laboratório Lewis por fornecerem comentários construtivos e sugestões sobre o manuscrito. A pesquisa sobre imunidade vegetal no laboratório de Lewis foi apoiada pelo USDA ARS 2030-21000-046-00D e 2030-21000-050-00D (JDL), e pela Diretoria de Ciências Biológicas iOS-1557661 (JDL).
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Materiais
| Nome | Empresa | Número de catálogo | Comentários |
|---|---|---|---|
| Fita 3M Micropore 1/2" x 10 YD CS 240 (1,25 cm x 9,1 m) | VWR International | 56222-182 | |
| Esferas de vidro borossilicato de 3 mm | Friedrich & Dimmock | GB3000B | |
| Bacto peptona | BD | 211677 | |
| Bacto ágar | BD | 214010 | |
| Biofotômetro Plus | Eppendorf | E952000006 | |
| Gabinete de biossegurança, classe II tipo A2 | |||
| MARCA Cuvetes de plástico descartáveis, poliestireno | VWR International | 47744-642 | |
| Palitos de dente de madeira lisa de Chenille | Kraft VWR International | 500029-808 | |
| cicloheximida | Research Products | International C81040-5.0 | |
| Fosfato de potássio dibásico anidro, grau ACS | |||
| Etanol - 190 Prova | |||
| Falcon poliestireno 96 poços microplacas, fundo plano | Fisher Scientific | 08-772-3 | |
| Queimador de Álcool de Vidro Wick Fisher | Scientific | S41898A / No. W-125 | |
| Queimadores de Álcool de Vidro | Fisher Scientific | S41898 / No. BO125 | |
| Glicerol Reagente ACS | VWR International | EMGX0185-5 | |
| Kimberly-Clark™ Kimtech Science™ Kimwipes™ Limpadores de Tarefas Delicadas | Fisher Scientific | 06-666-A | |
| Cloreto de magnésio, grau ACS | VWR International | 97061-356 | |
| Sulfato de magnésio heptahidratado, grau ACS | VWR International | 97062-130 | |
| Tubos de microcentrífuga, Tubos | |||
| 2,2 mL | |||
| , 115 volts | Bio Spec Products Inc. | 1001 | |
| Murashige & Skoog, Sais Basais | Caisson Laboratories, Inc. | MSP01-50LT | |
| Pipet-Lite XLS LTS 8-CH Pipet 20-200uL | Rainin | L8-200XLS | |
| Pipet-Lite XLS LTS 8-CH Pipet 2-20uL | Rainin | L8-20XLS | |
| Poliestireno 100mm x 25mm placa de Petri estéril | VWR International | 89107-632 | |
| Placa de Petri estéril de poliestireno 150mm x 15mm | Fisher Scientific | FB08-757-14 | |
| Placa de Petri estéril de poliestireno 150x15mm | Fisher Scientific | 08-757-148 | |
| Pure Bright Germicidal Ultra Bleach 5,7% Cloro Disponível (definido como 100% alvejante) | Staples | 1013131 | |
| Rifampicin | Gold Biotechnology | R-120-25 | |
| Silwet L-77 (copolímero de surfactante organosilicone não iônico C13H34O4 Si3 surfactante) | Fisher Scientific | NCO138454 | |
| Tips LTS 20 μ L 960/10 GPS-L10 | Rainin | 17005091 | |
| Pontas LTS 250 μ L 960/10 GPS-L250 | Rainin | 17005093 | |
| pinça de dissecação VWR ponta fina, 4,5" | VWR International | 82027-386 |
Referências
- Underwood, W., Melotto, M., He, S. Y. Role of plant stomata in bacterial invasion. Cell Microbiology. 9 (7), 1621-1629 (2007).
- Zipfel, C. Early molecular events in PAMP-triggered immunity. Current Opinion in Plant Biology<....
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Reimpressões e permissões
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Formal Correction: Erratum: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay
Posted by JoVE Editors on 10/18/2023. Citeable Link.
An erratum was issued for: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay. The Introduction, Protocol, Representative Results and Discussion sections were updated.
The last paragraph of the Introduction section was updated from:
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7–14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain T1 (PstT1), and can easily be adapted to other bacterial strains of interest.
to:
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7–14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain 19 (Pst19), and can easily be adapted to other bacterial strains of interest.
Step 4.8 of the Protocol section was updated from:
- After 1 h, transfer the bottle to the biosafety cabinet and under aseptic conditions, add 1,600 µL of sterile 1 M MgSO4, and appropriate antibiotics to the media.
NOTE: For rifampicin resistant strains PstDC3000 and PstT1, use rifampicin dissolved in dimethylformamide at a final concentration of 50 µg/mL. Use cycloheximide dissolved in ethanol at a final concentration of 50 µg/mL to prevent fungal growth on the plates.
to:
- After 1 h, transfer the bottle to the biosafety cabinet and under aseptic conditions, add 1,600 µL of sterile 1 M MgSO4, and appropriate antibiotics to the media.
NOTE: For rifampicin resistant strains PstDC3000 and Pst19, use rifampicin dissolved in dimethylformamide at a final concentration of 50 µg/mL. Use cycloheximide dissolved in ethanol at a final concentration of 50 µg/mL to prevent fungal growth on the plates.
Step 5.2 of the Protocol section was updated from:
- Patch bacteria (i.e., PstT1) from a glycerol stock onto KB agar with appropriate antibiotics (section 4).
to:
- Patch bacteria (i.e., Pst19) from a glycerol stock onto KB agar with appropriate antibiotics (section 4).
Step 5.6 of the Protocol section was updated from:
- For PstT1, incubate the KB plate at 28 ˚C for 48 h prior to using bacteria in the flood experiment.
to:
- For Pst19, incubate the KB plate at 28 ˚C for 48 h prior to using bacteria in the flood experiment.
Step 6 of the Protocol section was updated from:
6. Preparation of PstT1 inoculum
to
6. Preparation of Pst19 inoculum
Step 6.2 of the Protocol section was updated from:
- Perform serial dilutions using sterile 10 mM MgCl2 solution in the biosafety cabinet. For PstT1, use a spectrophotometer to make inoculum with a starting concentration of OD600 = 0.1.
to:
- Perform serial dilutions using sterile 10 mM MgCl2 solution in the biosafety cabinet. For Pst19, use a spectrophotometer to make inoculum with a starting concentration of OD600 = 0.1.
Step 6.3 of the Protocol section was updated from:
- For PstT1, make a 1/10 dilution from the initial resuspension at OD600 = 0.1 to obtain a serial dilution at a concentration of OD600 = 0.01.
to:
- For Pst19, make a 1/10 dilution from the initial resuspension at OD600 = 0.1 to obtain a serial dilution at a concentration of OD600 = 0.01.
Step 8.3 of the Protocol section was updated from:
- Set a timer for 3 min. Measure 6 mL of final inoculum (PstT1 OD600 = 0.0075 [section 6] or PstDC3000 OD600 = 0.005 [section 7]) and transfer 6 mL of inoculum to each plate with the 10-day-old seedlings.
to:
- Set a timer for 3 min. Measure 6 mL of final inoculum (Pst19 OD600 = 0.0075 [section 6] or PstDC3000 OD600 = 0.005 [section 7]) and transfer 6 mL of inoculum to each plate with the 10-day-old seedlings.
Step 8.11 of the Protocol section was updated from:
- Phenotype after 7–10 days for PstDC3000 or 10–14 days for PstT1 (section 11). If carrying out bacterial growth assays, collect leaf tissue after 4 days (sections 9 and 10) and then phenotype (section 11). Alternatively, perform phenotypic analysis and bacterial growth assays on separate sets of plants.
to:
- Phenotype after 7–10 days for PstDC3000 or 10–14 days for Pst19 (section 11). If carrying out bacterial growth assays, collect leaf tissue after 4 days (sections 9 and 10) and then phenotype (section 11). Alternatively, perform phenotypic analysis and bacterial growth assays on separate sets of plants.
Step 10.7 of the Protocol section was updated from:
- After obtaining colony counts (Figure 2B), normalize the counts to 0.01 g of tissue for seedlings and convert to log bacterial growth (Table 1).
NOTE: The average mass of one Moneymaker-PtoS cotyledon is 0.01 g and is empirically determined22.
| Genotype1 Column A | Tissue Weight (g) Column B | # of Colonies in a spot Column C | Dilution factor for spot2 Column D | Adjusted # of Colonies3 Column E | Dilution factor for serial dilution Column F | Total # of Colonies Column G (cfu/0.01 g)4 | Average # of Colonies (cfu/0.01 g) Column H | Average Log Growth (cfu/0.01 g (log10)) Column I |
| Sample 1 | 0.004 g | 10 | 200 | calculated as: (C2 x 0.01 g) / B2 = 25 | 1000 | calculated as: (D2 x E2 x F2) = 5000000 | average for sample 1 through last sample: (ie. average G1:G3) = 7000000 | log of average ie. log(H2) = 6.85 |
| Sample 2 | 0.003 g | 15 | 200 | 50 | 1000 | 10000000 | ||
| Sample 3 | 0.002 g | 6 | 200 | 30 | 1000 | 6000000 | ||
| 1Data shown for 3 samples | ||||||||
| 2Based on plating 5 µL x 200 for 1 mL | ||||||||
| 3Cotyledons are too small to core so colony counts were normalized to 0.01 g of tissue based on the average mass of one MoneyMaker-PtoS cotyledon (data not shown) | ||||||||
| 4Adjusted per mL based on volume plated | ||||||||
Table 1: Sample calculations for seedling bacterial growth assay. Sample calculations demonstrate how to normalize bacterial counts and determine log bacterial growth.
to:
- After obtaining colony counts (Figure 2B), normalize the counts to 0.1 g of tissue for seedlings and convert to log bacterial growth (Table 1).
NOTE: The average mass of one Moneymaker-PtoS cotyledon is 0.1 g and is empirically determined22.
| Genotype1 Column A | Tissue Weight (g) Column B | # of Colonies in a spot Column C | Dilution factor for spot2 Column D | Adjusted # of Colonies3 Column E | Dilution factor for serial dilution Column F | Total # of Colonies Column G (cfu/0.01 g)4 | Average # of Colonies (cfu/0.01 g) Column H | Average Log Growth (cfu/0.1 g (log10)) Column I |
| Sample 1 | 0.04 g | 10 | 200 | calculated as: (C2 x 0.1 g) / B2 = 25 | 1000 | calculated as: (D2 x E2 x F2) = 5000000 | average for sample 1 through last sample: (ie. average G1:G3) = 7000000 | log of average ie. log(H2) = 6.85 |
| Sample 2 | 0.03 g | 15 | 200 | 50 | 1000 | 10000000 | ||
| Sample 3 | 0.02 g | 6 | 200 | 30 | 1000 | 6000000 | ||
| 1Data shown for 3 samples | ||||||||
| 2Based on plating 5 µL x 200 for 1 mL | ||||||||
| 3Cotyledons are too small to core so colony counts were normalized to 0.1 g of tissue based on the average mass of one MoneyMaker-PtoS cotyledon (data not shown) | ||||||||
| 4Adjusted per mL based on volume plated | ||||||||
Table 1: Sample calculations for seedling bacterial growth assay. Sample calculations demonstrate how to normalize bacterial counts and determine log bacterial growth.
Step 11.3 of the Protocol section was updated from:
- Phenotype plants infected with PstT1 at 10–14 days after flood inoculation.
to:
- Phenotype plants infected with Pst19 at 10–14 days after flood inoculation.
Figure 4 in the Protocol section was updated from:
Figure 4: Schematic representation of expected phenotypes for seedling resistance and death in various genetic backgrounds. (A) Seedlings of Rio Grande-PtoR and the near-isogenic cultivar Rio Grande-PtoS are displayed 7 days after flooding with PstDC3000 (OD600 = 0.005) + 0.015% surfactant. Rio Grande-PtoR displays consistent resistance, and Rio Grande-PtoS displays consistent susceptibility to infection with PstDC3000. These lines give rise to discrete and binary phenotypes. (B) Seedlings of a wild accession, such as Solanum neorickii LA1329, are shown 10 days after flooding with PstT1 (OD600 = 0.0075) + 0.015% surfactant. Seedlings display phenotypic variability but were recorded as binary phenotypes. The amount of phenotypic variability and the method of phenotyping (binary resistance or resistance spectrum) will depend on the particular accession tested. (C) Mapping populations generated by outcrossing wild accessions to susceptible cultivars may display a wider spectrum of phenotypes in F2 segregating populations. In this case, it may be most appropriate to record seedling phenotypes on a spectrum. Highly susceptible seedlings from a mapping population may be phenotyped for death as early as day 7 when flooded with PstT1, and typically show a brown apical meristem, no to very little extension of the epicotyl, and no new, green vegetative growth. The apical meristem of susceptible seedlings may stay green or very light brown for more time, and there may be some extension of the epicotyl and very little vegetative growth, which turns brown and arrests by day 10. Individual seedlings can be phenotyped for resistance based on the amount of new and ongoing vegetative growth by day 14. Seedlings can then be grouped based on the phenotypes described above into different categories of resistance such as weak, medium, or strong resistance. Please click here to view a larger version of this figure.
to:
Figure 4: Schematic representation of expected phenotypes for seedling resistance and death in various genetic backgrounds. (A) Seedlings of Rio Grande-PtoR and the near-isogenic cultivar Rio Grande-PtoS are displayed 7 days after flooding with PstDC3000 (OD600 = 0.005) + 0.015% surfactant. Rio Grande-PtoR displays consistent resistance, and Rio Grande-PtoS displays consistent susceptibility to infection with PstDC3000. These lines give rise to discrete and binary phenotypes. (B) Seedlings of a wild accession, such as Solanum neorickii LA1329, are shown 10 days after flooding with Pst19 (OD600 = 0.0075) + 0.015% surfactant. Seedlings display phenotypic variability but were recorded as binary phenotypes. The amount of phenotypic variability and the method of phenotyping (binary resistance or resistance spectrum) will depend on the particular accession tested. (C) Mapping populations generated by outcrossing wild accessions to susceptible cultivars may display a wider spectrum of phenotypes in F2 segregating populations. In this case, it may be most appropriate to record seedling phenotypes on a spectrum. Highly susceptible seedlings from a mapping population may be phenotyped for death as early as day 7 when flooded with Pst19, and typically show a brown apical meristem, no to very little extension of the epicotyl, and no new, green vegetative growth. The apical meristem of susceptible seedlings may stay green or very light brown for more time, and there may be some extension of the epicotyl and very little vegetative growth, which turns brown and arrests by day 10. Individual seedlings can be phenotyped for resistance based on the amount of new and ongoing vegetative growth by day 14. Seedlings can then be grouped based on the phenotypes described above into different categories of resistance such as weak, medium, or strong resistance. Please click here to view a larger version of this figure.
The second paragraph of the Representative Results section was updated from:
Phenotypic screening of wild accessions using the seedling resistance assay
Figure 6 shows representative results for seedlings of susceptible and resistant accessions 10–14 days after flooding with PstT1. Susceptible accessions include RG-PtoR, S. pimpinellifolium LA1375, and S. pimpinellifolium LA1606, and resistant accessions include S. neorickii LA1329. Ten-day-old seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control, and PstT1 at an optical density of 0.0075 + 0.015% surfactant. The seedlings were phenotyped at least 10 days after flooding, as PstT1-infected seedlings died more slowly than PstDC3000-infected seedlings. Mock-inoculated seedlings were green, healthy, and actively growing. This control is important to ensure that the accessions are not sensitive to the concentration of surfactant, and to ensure there is no bacterial contamination. Susceptible accessions (Rio Grande-PtoR [n = 7], S. pimpinellifolium LA1375 [n = 7], and S. pimpinellifolium LA1606 [n = 5]) were dead, had brown apical meristems, and lacked new growth 10–14 days after inoculation with PstT1. In contrast, two S. neorickii LA1329 (n = 3) seedlings displayed a high level of new, green growth and survived infection with PstT1 (Figure 6). Three LA1329 seedlings did not germinate. Typically, 5–7 individuals were screened for each accession in a primary screen to determine the prevalence of resistance in the population. When a more genetically complex wild accession, such as LA1329, is flooded with PstT1, the resistance phenotypes display slightly more variability among individual seedlings, compared to Moneymaker-PtoR treated with PstDC3000. However, the resistance phenotypes were usually less variable than those seen in F2 mapping populations. Thus, binary phenotyping criteria was used for LA1329.
to:
Phenotypic screening of wild accessions using the seedling resistance assay
Figure 6 shows representative results for seedlings of susceptible and resistant accessions 10–14 days after flooding with Pst19. Susceptible accessions include RG-PtoR, S. pimpinellifolium LA1375, and S. pimpinellifolium LA1606, and resistant accessions include S. neorickii LA1329. Ten-day-old seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control, and Pst19 at an optical density of 0.0075 + 0.015% surfactant. The seedlings were phenotyped at least 10 days after flooding, as Pst19-infected seedlings died more slowly than PstDC3000-infected seedlings. Mock-inoculated seedlings were green, healthy, and actively growing. This control is important to ensure that the accessions are not sensitive to the concentration of surfactant, and to ensure there is no bacterial contamination. Susceptible accessions (Rio Grande-PtoR [n = 7], S. pimpinellifolium LA1375 [n = 7], and S. pimpinellifolium LA1606 [n = 5]) were dead, had brown apical meristems, and lacked new growth 10–14 days after inoculation with Pst19. In contrast, two S. neorickii LA1329 (n = 3) seedlings displayed a high level of new, green growth and survived infection with Pst19 (Figure 6). Three LA1329 seedlings did not germinate. Typically, 5–7 individuals were screened for each accession in a primary screen to determine the prevalence of resistance in the population. When a more genetically complex wild accession, such as LA1329, is flooded with Pst19, the resistance phenotypes display slightly more variability among individual seedlings, compared to Moneymaker-PtoR treated with PstDC3000. However, the resistance phenotypes were usually less variable than those seen in F2 mapping populations. Thus, binary phenotyping criteria was used for LA1329.
Figure 6 in the Representative Results section was updated from:
Figure 6: Phenotypic characterization of resistance or disease symptoms 10–14 days post-infection in wild accessions. Rio Grande-PtoR, S. pimpinellifolium LA1606, S. pimpinellifolium LA1375 and S. neorickii LA1329 tomato seedlings were grown on 0.5x MS plates for 10 days, and then flooded with PstT1 (OD600 = 0.0075) + 0.015% surfactant. The number of surviving seedlings for each wild accession out of the total number tested is shown. Scale bar = 1 cm. Please click here to view a larger version of this figure.
to:
Figure 6: Phenotypic characterization of resistance or disease symptoms 10–14 days post-infection in wild accessions. Rio Grande-PtoR, S. pimpinellifolium LA1606, S. pimpinellifolium LA1375 and S. neorickii LA1329 tomato seedlings were grown on 0.5x MS plates for 10 days, and then flooded with Pst19 (OD600 = 0.0075) + 0.015% surfactant. The number of surviving seedlings for each wild accession out of the total number tested is shown. Scale bar = 1 cm. Please click here to view a larger version of this figure.
The third paragraph of the Representative Results section was updated from:
Quantitative assessment of bacterial growth using the seedling flood assay
To confirm that the observed resistance in LA1329 to PstT1 resulted in lower bacterial growth, bacterial growth assays were carried out in tomato seedlings. The level of PstT1 growth in Moneymaker-PtoS and S. neorickii LA1329 was determined 4 days post-infection. Moneymaker-PtoS is a near-isogenic line with consistent susceptibility among individual seedlings. Wild accessions such as S. neorickii LA1329 are often more genetically complex. LA1329 displays approximately 60% resistance to PstT1 across the population22. Because seedlings may drop their cotyledons after infection, one seedling was grown on each plate to correlate bacterial growth in the harvested cotyledon with overall seedling survival or death as determined phenotypically at least 10 days after flooding. The bacterial counts on day 4 for each seedling were normalized to 0.01 g of tissue and converted to log growth (CFU/0.01 g(log10)). Log growth for phenotypically resistant LA1329 seedlings (LA1329RES) or phenotypically susceptible seedlings (LA1329SUS) were separately pooled and compared to each other and the susceptible cultivar Moneymaker-PtoS. For example, there was a 1.7 log difference in bacterial growth between LA1329RES (log 6.3) and LA1329SUS (log 8.0), and a 1.6 log difference between LA1329RES (log 6.3) and Moneymaker-PtoS (log 7.9) (Figure 7). Therefore, phenotypic resistance correlated with quantitative resistance in the seedling assays.
to:
Quantitative assessment of bacterial growth using the seedling flood assay
To confirm that the observed resistance in LA1329 to Pst19 resulted in lower bacterial growth, bacterial growth assays were carried out in tomato seedlings. The level of Pst19 growth in Moneymaker-PtoS and S. neorickii LA1329 was determined 4 days post-infection. Moneymaker-PtoS is a near-isogenic line with consistent susceptibility among individual seedlings. Wild accessions such as S. neorickii LA1329 are often more genetically complex. LA1329 displays approximately 60% resistance to Pst19 across the population22. Because seedlings may drop their cotyledons after infection, one seedling was grown on each plate to correlate bacterial growth in the harvested cotyledon with overall seedling survival or death as determined phenotypically at least 10 days after flooding. The bacterial counts on day 4 for each seedling were normalized to 0.01 g of tissue and converted to log growth (CFU/0.01 g(log10)). Log growth for phenotypically resistant LA1329 seedlings (LA1329RES) or phenotypically susceptible seedlings (LA1329SUS) were separately pooled and compared to each other and the susceptible cultivar Moneymaker-PtoS. For example, there was a 1.7 log difference in bacterial growth between LA1329RES (log 6.3) and LA1329SUS (log 8.0), and a 1.6 log difference between LA1329RES (log 6.3) and Moneymaker-PtoS (log 7.9) (Figure 7). Therefore, phenotypic resistance correlated with quantitative resistance in the seedling assays.
Figure 7 in the Representative Results section was updated from:
x
Figure 7: Resistant Solanum neorickii LA1329 seedlings support lower bacterial growth than Moneymaker-PtoS or susceptible S. neorickii LA1329. Bacterial counts were determined 4 days post-inoculation from S. neorickii LA1329 (n = 14) and Moneymaker-PtoS (n = 10) seedlings infected with PstT1 and normalization was performed to 0.01 g of tissue. For LA1329, the two phenotypic groups, susceptible (SUS) or resistant (RES), were observed and counted separately. Above the bar * = statistically significant difference determined by a one-factor analysis of variance. A general linear model procedure (p < 0.001) followed by a multiple comparison of means using Tukey's post hoc test was used. Error bars = standard error. The figure indicates one representative experiment. Please click here to view a larger version of this figure.
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Figure 7: Resistant Solanum neorickii LA1329 seedlings support lower bacterial growth than Moneymaker-PtoS or susceptible S. neorickii LA1329. Bacterial counts were determined 4 days post-inoculation from S. neorickii LA1329 (n = 14) and Moneymaker-PtoS (n = 10) seedlings infected with Pst19 and normalization was performed to 0.1 g of tissue. For LA1329, the two phenotypic groups, susceptible (SUS) or resistant (RES), were observed and counted separately. Above the bar * = statistically significant difference determined by a one-factor analysis of variance. A general linear model procedure (p < 0.001) followed by a multiple comparison of means using Tukey's post hoc test was used. Error bars = standard error. The figure indicates one representative experiment. Please click here to view a larger version of this figure.
The first paragraph of the Discussion section was updated from:
A protocol for flood inoculation with PstDC3000 or PstT1 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf cluster and complete death on a susceptible cultivar lacking the Pto/Prf cluster22. For a strain such as PstT1, where there are no known resistant varieties, the optical density was optimized to be the lowest possible for consistent and complete plant death22. Uppalapati et al.24 designed a tomato seedling assay to investigate the pathogenesis of PstDC3000 and the virulence function of coronatine. In this virulence assay, infections were performed using bacteria concentrated to an OD600 of 0.124, 20x higher than the optical density of strains used in our resistance assay. Recognition of PstDC3000 effectors AvrPto and AvrPtoB in tomato seedlings carrying the Pto/Prf gene cluster results in ETI and a macroscopic HR22. In the context of a strong immune response such as ETI, a lower bacterial titer was used for PstDC3000 to avoid overwhelming genetic resistance from the Pto/Prf gene cluster22. In addition, these results suggest that a high bacterial concentration could overwhelm weaker immune responses such as PTI or quantitative partial resistance, where multiple genes contribute to the overall phenotype. Surfactant is necessary for the bacteria to adhere to the leaf surface; however, high concentrations can cause chlorosis of the leaf22. We previously tested a range of surfactant concentrations to empirically determine the ideal concentration in 10-day-old tomato seedlings22. When testing new species that may differ in their sensitivity to surfactant, the surfactant concentration should be optimized to identify a concentration that does not cause damage or chlorosis in the absence of bacteria. Appropriate assay conditions will require optimization of a surfactant concentration that does not cause damage, and a bacterial concentration that causes disease in all susceptible controls.
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A protocol for flood inoculation with PstDC3000 or Pst19 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf cluster and complete death on a susceptible cultivar lacking the Pto/Prf cluster22. For a strain such as Pst19, where there are no known resistant varieties, the optical density was optimized to be the lowest possible for consistent and complete plant death22. Uppalapati et al.24 designed a tomato seedling assay to investigate the pathogenesis of PstDC3000 and the virulence function of coronatine. In this virulence assay, infections were performed using bacteria concentrated to an OD600 of 0.124, 20x higher than the optical density of strains used in our resistance assay. Recognition of PstDC3000 effectors AvrPto and AvrPtoB in tomato seedlings carrying the Pto/Prf gene cluster results in ETI and a macroscopic HR22. In the context of a strong immune response such as ETI, a lower bacterial titer was used for PstDC3000 to avoid overwhelming genetic resistance from the Pto/Prf gene cluster22. In addition, these results suggest that a high bacterial concentration could overwhelm weaker immune responses such as PTI or quantitative partial resistance, where multiple genes contribute to the overall phenotype. Surfactant is necessary for the bacteria to adhere to the leaf surface; however, high concentrations can cause chlorosis of the leaf22. We previously tested a range of surfactant concentrations to empirically determine the ideal concentration in 10-day-old tomato seedlings22. When testing new species that may differ in their sensitivity to surfactant, the surfactant concentration should be optimized to identify a concentration that does not cause damage or chlorosis in the absence of bacteria. Appropriate assay conditions will require optimization of a surfactant concentration that does not cause damage, and a bacterial concentration that causes disease in all susceptible controls.
The third paragraph of the Discussion section was updated from:
Pst is a foliar pathogen that preferentially colonizes the aerial parts of tomato seedlings, including the cotyledons24 (Figure 3). Therefore, qualitative phenotyping in the seedling flood assay focuses on growth and disease symptoms in aerial portions of the seedling, and tissue for the bacterial growth assay is sampled from the cotyledons for quantitative analysis. After flood inoculation, seedlings may die within 7–10 days after inoculation with PstDC3000 or 10–14 days after inoculation with PstT1, as discussed in section 11. Seedling death is visualized by a brown apical meristem, arrested epicotyl elongation, and/or arrested vegetative growth. If different bacterial strains are used, the timing will have to be empirically determined. In addition, the progression of disease on control plants should be monitored daily after flooding until a consistent time frame from the onset of disease symptoms to seedling death can be identified. Depending on the genotypes and treatments used in the flood assay, seedling phenotypes can be recorded as binary phenotypes or on a disease spectrum (Figure 4). A broader spectrum of phenotypes may be observed when flood inoculating F2 mapping populations from wild tomato accessions crossed to susceptible cultivars (Figure 4C). It may be best to phenotype segregating populations on a disease spectrum depending on how quickly the seedling dies and the degree of new vegetative growth and branching (Figure 4C). The seedling flood assay can also be used in conjunction with the seedling bacterial growth assay to quantitatively assess levels of bacterial growth associated with qualitative phenotypes in individual seedlings (Figure 7). Very large reductions (i.e., ~log 3) in bacterial growth or strong resistance in resistant seedlings of a wild accession compared to a susceptible cultivar suggest that the underlying genetic basis of resistance may be due to ETI22. Smaller reductions in bacterial growth (i.e., ~log 1.7), as observed in LA1329 seedlings, may be due to the contribution of weaker resistance from quantitative trait loci and/or PTI. Thus, the seedling growth assay can be an important tool in further characterizing resistance in wild tomato lines.
to:
Pst is a foliar pathogen that preferentially colonizes the aerial parts of tomato seedlings, including the cotyledons24 (Figure 3). Therefore, qualitative phenotyping in the seedling flood assay focuses on growth and disease symptoms in aerial portions of the seedling, and tissue for the bacterial growth assay is sampled from the cotyledons for quantitative analysis. After flood inoculation, seedlings may die within 7–10 days after inoculation with PstDC3000 or 10–14 days after inoculation with Pst19, as discussed in section 11. Seedling death is visualized by a brown apical meristem, arrested epicotyl elongation, and/or arrested vegetative growth. If different bacterial strains are used, the timing will have to be empirically determined. In addition, the progression of disease on control plants should be monitored daily after flooding until a consistent time frame from the onset of disease symptoms to seedling death can be identified. Depending on the genotypes and treatments used in the flood assay, seedling phenotypes can be recorded as binary phenotypes or on a disease spectrum (Figure 4). A broader spectrum of phenotypes may be observed when flood inoculating F2 mapping populations from wild tomato accessions crossed to susceptible cultivars (Figure 4C). It may be best to phenotype segregating populations on a disease spectrum depending on how quickly the seedling dies and the degree of new vegetative growth and branching (Figure 4C). The seedling flood assay can also be used in conjunction with the seedling bacterial growth assay to quantitatively assess levels of bacterial growth associated with qualitative phenotypes in individual seedlings (Figure 7). Very large reductions (i.e., ~log 3) in bacterial growth or strong resistance in resistant seedlings of a wild accession compared to a susceptible cultivar suggest that the underlying genetic basis of resistance may be due to ETI22. Smaller reductions in bacterial growth (i.e., ~log 1.7), as observed in LA1329 seedlings, may be due to the contribution of weaker resistance from quantitative trait loci and/or PTI. Thus, the seedling growth assay can be an important tool in further characterizing resistance in wild tomato lines.
The fourth paragraph of the Discussion section was updated from:
Typically, genetic screens have been performed on four- to five-week-old adult tomato plants to identify the genetic basis of P. syringae resistance in wild accessions20,21. Adult tomato plants require much longer growth times, require more space in the growth chamber, and are much larger plants, which means that usually few individuals are screened for each line. The seedling flood assay provides a powerful, alternative approach in the identification of P. syringae resistance in wild tomato accessions. Screening at the seedling stage permits a large sample size to be tested which can be particularly advantageous in detecting resistance in genetically complex populations. Reduced growth chamber space requirements and growth time facilitate a high-throughput approach and rapid detection of natural resistance in wild accessions to emerging pathogens. Furthermore, P. syringae resistance that was identified at the seedling stage in this assay is not restricted to the developmental stage. S. neorickii LA1329 and S. habrochaites LA1253 were initially identified at the seedling stage and also display resistance to PstT1 in adult plants as previously described22.
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Typically, genetic screens have been performed on four- to five-week-old adult tomato plants to identify the genetic basis of P. syringae resistance in wild accessions20,21. Adult tomato plants require much longer growth times, require more space in the growth chamber, and are much larger plants, which means that usually few individuals are screened for each line. The seedling flood assay provides a powerful, alternative approach in the identification of P. syringae resistance in wild tomato accessions. Screening at the seedling stage permits a large sample size to be tested which can be particularly advantageous in detecting resistance in genetically complex populations. Reduced growth chamber space requirements and growth time facilitate a high-throughput approach and rapid detection of natural resistance in wild accessions to emerging pathogens. Furthermore, P. syringae resistance that was identified at the seedling stage in this assay is not restricted to the developmental stage. S. neorickii LA1329 and S. habrochaites LA1253 were initially identified at the seedling stage and also display resistance to Pst19 in adult plants as previously described22.