Yöntem makalesi

Pseudomonas syringae pv Direnci yüksek-Throughput Tanımlama. Fide Sel Asası Kullanarak Domates

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DOI:

10.3791/60805

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10 Mart 2020

 ,  , 
1Department of Plant and Microbial Biology, University of California, Berkeley, 2Plant Gene Expression Center, United States Department of Agriculture

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Düzeltme bildirimi

Important: There has been an erratum issued for this article. View Erratum Notice

Özet

Fide sel testi Pseudomonas syringae bakteridirenç için yabani domates katılımları hızlı tarama kolaylaştırır. Bu tetki, fide bakteri büyüme testi ile birlikte kullanılan, daha fazla bakteriye altta yatan direnç karakterize yardımcı olabilir, ve direnç genetik temelini belirlemek için popülasyonları tarama için kullanılabilir.

Özet

Domates Pseudomonas syringaetarafından enfekte edilebilir bir tarımsal olarak önemli bir üründür , Bir Gram-negatif bakteri, bakteriyel benek hastalığı ile sonuçlanan. Domates-P. syringae pv. domates patosistemi yaygın bitki doğuştan gelen tepkiler ve hastalık direnci genetik temelini incelemek için kullanılır. Hastalık, Solanum pimpinellifolium'dan Ekili domatese Pto/Prf gen kümesinin getirilmesiyle uzun yıllar boyunca başarılı bir şekilde yönetilirken, P. syringae'nin 1.

Yabani domates türleri patojen tanımada doğal çeşitliliğin önemli rezervuarlarıdır, çünkü farklı patojen basınçları ile farklı ortamlarda evrimleşmişlerdir. Yabani domates hastalık direnci için tipik ekranlarda, yetişkin bitkiler, onların uzun büyüme süresi ve daha fazla büyüme alanı gereksinimleri nedeniyle taranabilir bitki sayısını sınırlayabilir kullanılır. 10 günlük domates fidelerini direnç açısından taramak için bir yöntem geliştirdik, bu yöntem bitki büyüme süresini ve büyüme odası alanını en aza indirir, bitkilerin hızlı bir şekilde devrilmesini sağlar ve büyük numune boyutlarının test edilmesine olanak sağlar. Hayatta kalma veya ölüm fide sonuçları ayrık fenotipler olarak veya sel sonrası kalan fidelerde yeni büyüme miktarı ile tanımlanan bir direnç ölçeğinde tedavi edilebilir. Bu yöntem, 10 günlük domates fidelerini iki P. syringae suşlarına karşı direnç açısından taramak için optimize edilmiştir ve diğer P. syringae suşlarına kolayca adapte edilebilir.

Giriş

Pseudomonas syringae, çok çeşitli bitki konaklarını enfekte eden gram-negatif patojenik bir bakteridir. Bakteriler stomata veya fiziksel yaralar yoluyla konak bitki girin ve apoplast1çoğalır. Bitkiler bakteriyel patojenler tarafından enfeksiyona karşı korumak için iki katmanlı bir bağışıklık yanıtı gelişti. Birinci seviye bitki hücre yüzeyinde oluşur, burada bitki hücre zarında desen tanıma reseptörleri son derece korunmuş patojen ilişkili moleküler desenler algılar (PAMPs) PAMP tetikli bağışıklık denilen bir süreç (PTI)2. Bu işlem sırasında, konak bitki hücre duvarına kalkoz birikimi, stomata kapatılması, reaktif oksijen türlerinin üretimi ve patogenez ile ilgili genlerin indüksiyon dahil olmak üzere savunma tepki yollarını düzenler.

Bakteriler, proteinler olarak adlandırılan proteinleri doğrudan bitki hücresi3'eulaştırmak için tip III salgı sistemini kullanarak PTI'nin üstesinden gelebilirler. Efektör proteinler genellikle PTI bileşenleri hedef ve patojen virülans teşvik4. Bitki bağışıklığının ikinci kademesi, etki alanı proteinlerin tanınması üzerine bitki hücresi içinde oluşur. Bu tanıma direnç genleri bağlıdır, hangi nükleotit bağlayıcı site lösin açısından zengin tekrar içeren reseptörleri (NLR). NLR'ler ya doğrudan efektörleri tanıyabilme ya da bir virülans hedefi ndeki aktivitelerini tanıma ya da5. Daha sonra etkili tetikli bağışıklık denilen bir süreçte ikincil bir bağışıklık yanıtı tetikler (ETI), genellikle aşırı duyarlı bir yanıt ile ilişkili (İK), enfeksiyon yerinde lokalize hücre ölümü bir formu6. ETI ile ilişkili gen-gen direncinin aksine, bitkiler birden fazla genin katkısına bağlı olan kantitatif kısmi dirençgösterebilirler 7.

P. syringae pv. domates (Pst)domates bakteriyel benek nedensel ajan dır ve kalıcı bir tarımsorunudur. Alanında baskın suşları genellikle Pst yarış 0 suşları ya da tip III efektörleri AvrPto ve AvrPtoB her ikisini ifade edilmiştir. DC3000(PstDC3000) temsili bir ırk 0 suşu ve domates bakteriyel leke neden olabilir bir model patojen. Bakteriyel benek hastalığı ile mücadele etmek için, yetiştiriciler Pto introgressed [P. syringae pv. domates]/ Prf [Pto direnci ve fenthion duyarlılık] yabani domates türlerinden gen kümesi Solanum pimpinellifolium içine modern çeşitleri8,9. Pto geni, Prf NLR ile birlikte AvrPto ve AvrPtoB10,11,12,13,14efektörleri tanınması yoluyla PstDC3000 direnç vermek bir serin-threonine protein kimyaz kodlar . Ancak, bu direnç son yıllarda onların hızlı ve agresif yayılması için izin, ortaya çıkan Yarış 1 suşları karşıetkisizdir15,16. AvrPto ya kayıp ya da bu suşları mutasyona uğramış çünkü Yarış 1 suşları, Pto / Prf küme tarafından tanıma kaçınmak, ve AvrPtoB minimal15birikir görünür,17,18.

Yabani domates popülasyonları Pst direnci için doğal varyasyon önemli rezervuarları ve daha önce potansiyel direnç loci belirlemek için kullanılmıştır19,20,21. Ancak, patojen direnci için mevcut ekranlar 4-5 haftalık yetişkin bitkiler20,21kullanır. Bu nedenle, büyüme süresi, büyüme odası alanı ve nispeten küçük örneklem boyutları ile sınırlıdır. Konvansiyonel yaklaşımların sınırlamalarını gidermek için, 10 günlük domates fideleri22kullanarak yüksek iş itimat lı domates P. syringae direnç tayini geliştirdik. Bu yaklaşım yetişkin bitkileri kullanma üzerinde çeşitli avantajlar sunar: yani, daha kısa büyüme süresi, azaltılmış alan gereksinimleri, ve daha yüksek iş artışı. Ayrıca, bu yaklaşımın erişkin bitkilerde gözlenen hastalık direnci fenotipleri sadakatle recapitulates göstermiştir22.

Bu protokolde açıklanan fide sel idamı, steril Murashige ve Skoog (MS) medyasının Petri kaplarında 10 gün boyunca yetiştirilir ve daha sonra ilgi ve yüzey aktif bakterisini içeren bir inokülle dolup taşmaktadır. Sel inden sonra, fideler bakteriyel büyüme tahlilleri ile hastalık direnci açısından nicel olarak değerlendirilebilir. Ayrıca, fide sağkalım veya ölüm sel sonra ayrı bir direnç veya hastalık fenotip 7-14 gün olarak hareket edebilir. Bu yaklaşım, Pst ırkı na karşı direnç için çok sayıda yabani domates katılımlarını tatmak için yüksek iş yapma alternatifi sunar, örneğin Pst suşu T1(PstT1) gibi, ve diğer bakteriyel suşlara kolayca adapte edilebilir.

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Protokol

1. Biyogüvenlik kabininin hazırlanması ve kullanımı

  1. Biyogüvenlik kabinini p etanolle silin.
  2. Kuşağı kapatın ve biyogüvenlik kabinindeki ultraviyole ışığı 15 dakika süreyle açın.
  3. 15 dakika sonra, biyogüvenlik kabinindeki ultraviyole ışığı kapatın. Kuşağı kaldırın ve üfleyiciyi 15 dk açın.
  4. Sterilize kabine öğeleri koymadan önce% 70 etanol ile biyogüvenlik kabininde kullanılacak tüm öğeleri silin.
  5. Biyogüvenlik kabininde çalışmadan önce eldivenleri veya çıplak elleri p etanolile temizleyin.
  6. Biyogüvenlik dolabının merkezinde, üfleyiciden uzakta çalışın.
  7. Deneyler için açılmamış otoklavlı steril 10 mM MgCl2 ve ultra saf H2O şişeler kullanın. Şişeleri biyogüvenlik dolabına koyun ve sadece sterilleştirilmiş biyogüvenlik kabininde açın, tezgahın üstüne değil.
  8. Sterilize biyogüvenlik kabininde çalışmak için özel cam pipetler ve pipet uçları kullanın. Bunların sadece biyogüvenlik kabininde açıldığından emin olun, asla tezgahın üstünde değil.
  9. Biyogüvenlik kabininin kullanımından sonra, tüm atıkları (çamaşır suyu atıkları hariç) otoklavla ve yüzeyi p etanol ile silin.

2. Bitki ortamının hazırlanması

  1. Tartmak ve ultrasaf H 0.5x MS bazal tuzları eritin2O. Tartmak 0.8% bacto agar ve sonra çözünmüş 0.5x MS ekleyin.
  2. Otoklav ve medya dökme veya borulama önce 1 saat için 50 °C su banyosunda soğumasını bekleyin.
  3. Plakaların aşırı doldurulmaması için, polistiren tek kullanımlık steril 100 x 25 mm plakaları 40 mL dolgu seviyesine işaretleyin. Sterilleştirilmiş biyogüvenlik kabininde 100 x 25 mm steril plakalara ortam dökün.

3. Bitki malzemelerinin hazırlanması ve büyüme koşulları

  1. Domates tohumlarını 2,2 mL mikrosentrifuge tüpüne yerleştirin ve 2,0 mLP çamaşır suyu çözeltisi ekleyin.
  2. 25 dakika boyunca bir rocker üzerinde tüp Rock.
  3. 25 dakika sonra, kayadan tohumları çıkarın ve steril biyogüvenlik kabininde bir pipet ile çamaşır suyu çözeltisi kaldırın. Tüm çamaşır suyunun çıkarıldığından emin olun.
  4. Tohumları yıkamak için 2 mL steril ultrasaf H2O ekleyin. Tüpü 5x ters çevir.
  5. Bir pipet ile tüp sıvı çıkarın.
  6. Tohumları 4kat daha fazla yıkamak için adımları 3.3-3.5 tekrarlayın.
  7. Steril ultrasafH2 O 2 mL ekleyin ve boş bir steril Petri kabına tohumları dökün.
  8. Etanoldeki alev büşremler ve 0,5x MS + %0,8 agar ortam içeren 100 x 25 mm plakalar üzerinde tohum aktarma ve eşit aralıklı tohumlar aktarmadan önce soğumasını sağlar.
  9. Bir plakanın ortasındabir çizgi de 5-7 tohum aktarın ve cerrahi bant (1,25 cm x 9,1 m) ile plakaların kenarlarını mühürleyin.
  10. Sterilize edilmiş tohumları en az 3 gün karanlıkta 4 °C'de senkronize edin ve çimlenmeyi senkronize edin. Tohumların tabakta kaymaması için plakaların düz ve yüz yukarı yığılmış olduğundan emin olun.
  11. Dikey olarak kökleri plaka yüzeyi boyunca aşağı büyüyecek şekilde plakaları yönlendirmek, tohum hattı yatay odaklı, büyüme odasına aktarırken.
    NOT: Büyüme odasını 22 °C'ye ayarlayın ve ~200-220 μE metre-2 s-1 ve 8 saat karanlık ışık yoğunluğunda 16 saat ışık sağlayın.
  12. Sel den önce, fidelerin genellikle tam olarak ortaya çıktığı ve genişletilmiş cotyledonları ve ortaya çıkan ilk gerçek yaprakları gösterdiği büyüme odasında 10 gün boyunca fideler yetiştirilir(Şekil 1).

figure-protocol-1
Şekil 1: Tipik 10 günlük domates fidelerinin gelişim evresi. Rio Grande-PtoR domates tohumları sterilize edildi, kaplandı ve 4 °C'de karanlıkta en az 3 gün boyunca tabakalaştırıldı. Fideler, 0.5x MS plakalar üzerinde 22 °C'de 10 gün boyunca su altında kalmadan önce yetiştirildi. Tipik olarak, 10 gün cotyledons tamamen genişletilmiş ve ilk gerçek yaprakları ortaya çıkmaya başlıyor. Bu rakamın daha büyük bir sürümünü görüntülemek için lütfen buraya tıklayın.

4. King's B23 (KB) ortamının hazırlanması

  1. 500 mL ultrapure H2O ile kabı doldurun ve bir karıştırma plakası üzerinde karıştırın.
  2. Tamamen çözünür 20 g bacto peptone, 1.5 g susuz K2HPO4, ve gliserol 12.5 mL ultrapure H2O ile bir kabın içinde.
  3. Çözünmüş karışımı 1 L'lik bir silindire dökün ve ultra saf H2O ile 1 L son hacmine kadar getirin.
  4. Suyu tekrar kabın içine dökün ve karıştırana kadar karıştırın.
  5. İki 500 mL cam şişe içine bacto agar 7,5 g tartın ve adım 4.4 her şişe içine KB suyu 500 mL ekleyin. 20 dk için otoklav.
  6. Otoklav dan şişe çıkarın ve yavaşça agar dağıtmak için girdap.
  7. Şişeleri 1 saat boyunca 50 °C'lik bir su banyosuna aktarın.
  8. 1 saat sonra, biyogüvenlik kabine ve aseptik koşullar altında şişe transferi, steril 1 M MgSO41.600 μL ekleyin , ve ortama uygun antibiyotikler.
    NOT: PstDC3000 ve PstT1 rifampisin dirençli suşları için, 50 μg/mL son konsantrasyonda dimetilformamid çözünmüş rifampisin kullanın. Plakalarda mantar büyümesini önlemek için 50 g/mL'lik son konsantrasyonda etanoliçinde çözünmüş siklohekamid kullanın.
  9. Karıştırmak için ortamı hafifçe döndürün ve tabakların altını kaplayacak şekilde dökün.
  10. Plakaların 4 °C'de baş aşağı saklamadan önce katılaşmaları için en az 1 saat bekleyin.

5. Bakteriyel suşların ve kültür koşullarının bakımı




figure-protocol-2


figure-protocol-3

figure-protocol-4

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Sonuçlar

Fide direnci tayini kullanılarak çeşitlerive isojenik hatlarda PtoRaracılı bağışıklığın saptanması
Şekil 5, PstDC3000 ile sel bastıktan 7-10 gün sonra Moneymaker-PtoR ve Moneymaker-PtoS çeşitleri için temsili sonuçlar gösterir. Enfeksiyondan önce, 10 günlük fideler tam olarak ortaya çıktı ve genişletilmiş cotyledons ve ortaya çıkan ilk gerçek yaprakları gösterdi. Fideler 10 mM MgCl2 + %0.015 yüzey aktif madde ile negatif kon...

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Tartışma

Domates fidelerinde bu bakteriyel suşlara karşı direnci tespit etmek için optimize edilen PstDC3000 veya PstT1 ile sel aşısı için bir protokol tanımlanmıştır. Fide direnci tahtında optimum sonuçlar için, ampirik olarak belirlenen bakteri konsantrasyonu ve yüzey aktif konsantrasyonu da dahil olmak üzere çeşitli kritik parametreler vardır22. PstDC3000 için optik yoğunluk, Pto/Prf kümesini içeren dirençli bir çeşitte tam hayatta kalma ve Pto/Prf kümesi

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Açıklamalar

Yazarların açıklayacak bir şeyi yok.

Teşekkürler

Biz hastalık veya direnç sonuçları üzerinde medya hacminin etkisini test için Jamie Calma teşekkür ederiz. Lewis Lab'dan Dr. Maël Baudin ve Dr. Karl J. Scheiber'a el yazması hakkında yapıcı yorum ve önerilerde bulunduklarından dolayı teşekkür ederiz. Lewis laboratuvarında bitki bağışıklığı üzerine yapılan araştırmalar USDA ARS 2030-21000-046-00D ve 2030-21000-050-00D (JDL) ve NSF Biyolojik Bilimler Müdürlüğü IOS-1557661 (JDL) tarafından desteklendi.

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Malzemeler

Bu makalede kullanılan malzemelerin listesi
AdŞirketKatalog numarasıYorumlar
3M Bant Mikro Gözenek 1/2" x 10 YD CS 240 (1,25 cm x 9,1 m)VWR International56222-182
3mm borosilikat cam boncuklarFriedrich & DimmockGB3000B
Bacto peptoneBD211677
Bacto agarBD214010
Biyofotometre PlusEppendorfE952000006
Biyogüvenlik kabini, sınıf II tip A2
MARKA Tek kullanımlık plastik küvetler, polistirenVWR International47744-642
Şönil Kraft Düz Ahşap KürdanVWR International500029-808
sikloheksimidAraştırma Ürünleri UluslararasıC81040-5.0
Dibazik potasyum fosfat susuz, ACS sınıfıFisher ScientificP288-500
Dimetilformamid
Diseksiyon mikroskobu (En az 10x büyütme)
Etanol - 190 Proof
Falcon polistiren 96 kuyulu mikroplakalar, düz tabanlıFisher Scientific08-772-3
Cam Alkol Brülör FitilFisher ScientificS41898A / No. W-125
Cam Alkol BrülörleriFisher ScientificS41898 / No. BO125
Gliserol ACS reaktifiVWR InternationalEMGX0185-5
Kimberly-Clark & trade; Kimtech Bilim ve Ticaret; Kimmendil ve ticaret; Hassas Görev SilecekleriFisher Scientific06-666-A
Magnezyum klorür, ACS sınıfıVWR Uluslararası97061-356
Magnezyum sülfat heptahidrat, ACS sınıfıVWR Uluslararası97062-130
Mikrosantrifüj tüpleri, 1.5 mL
Mikrosantrifüj tüpleri, 2.2 mL
Mini Boncuk Çırpıcı-96, 115 voltBio Spec Ürünler A.Ş.1001
Murashige & Skoog, Bazal Tuzlar Keson Laboratuvarları, Inc.MSP01-50LT
Pipet-Lite XLS LTS 8-CH Pipet 20-200uLRaininL8-200XLS
Pipet-Lite XLS LTS 8-CH Pipet 2-20uLRaininL8-20XLS
Polistiren 100mm x 25mm steril petri kabıVWR International89107-632
Polistiren 150mm x 15mm steril petri kabıFisher ScientificFB08-757-14
Polistiren 150x15mm steril petri kabıFisher Scientific08-757-148
Saf Parlak Antiseptik Ultra Ağartıcı %5,7 Mevcut Klor (%100 çamaşır suyu olarak tanımlanır)Zımbalar1013131
RifampisinAltın BiyoteknolojiR-120-25
Silwet L-77 (iyonik olmayan organosilikon yüzey aktif madde kopolimer C13H34O4 Si3 yüzey aktif madde)Fisher ScientificNCO138454
İpuçları LTS 20 & L 960/10 GPS-L10Rainin17005091
Uçları LTS 250 & mu; L 960/10 GPS-L250Rainin17005093
VWR diseksiyon forseps ince uç, 4.5"VWR International82027-386

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  19. Rose, L. E., Langley, C. H., Bernal, A. J., Michelmore, R. W. Natural variation in the Pto pathogen resistance gene within species of wild tomato (Lycopersicon). I. Functional analysis of Pto alleles. Genetics. 171 (1), 345-357 (2005).
  20. Thapa, S. P., Miyao, E. M., Davis, R. M., Coaker, G. Identification of QTLs controlling resistance to Pseudomonas syringae pv. tomato race 1 strains from the wild tomato Solanum habrochaites LA1777. Theoretical and Applied Genetics. 128 (4), 681-692 (2015).
  21. Bao, Z. L., et al. Identification of a candidate gene in Solanum habrochaites for resistance to a race 1 strain of Pseudomonas syringae pv. tomato. Plant Genome. 8 (3), 1-15 (2015).
  22. Hassan, J. A., Zhou, Y. J., Lewis, J. D. A rapid seedling resistance assay identifies wild tomato lines that are resistant to Pseudomonas syringae pv. tomato race 1. Molecular Plant-Microbe Interactions. 30 (9), 701-709 (2017).
  23. King, E. O., Ward, M. K., Raney, D. E. Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine. 44 (2), 301-307 (1954).
  24. Uppalapati, S. R., et al. Pathogenicity of Pseudomonas syringae pv. tomato on tomato seedlings: phenotypic and gene expression analyses of the virulence function of coronatine. Molecular Plant-Microbe Interactions. 21 (4), 383-395 (2008).
  25. Bhardwaj, V., Meier, S., Petersen, L. N., Ingle, R. A., Roden, L. C. Defence responses of Arabidopsis thaliana to infection by Pseudomonas syringae are regulated by the circadian clock. PLoS One. 6 (10), 26968(2011).
  26. Lu, H., McClung, C. R., Zhang, C. Tick tock: circadian regulation of plant innate immunity. Annual Review of Phytopathology. 55, 287-311 (2017).
  27. Wang, W., et al. Timing of plant immune responses by a central circadian regulator. Nature. 470 (7332), 110-114 (2011).

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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:

  1. 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:

  1. 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:

  1. Patch bacteria (i.e., PstT1) from a glycerol stock onto KB agar with appropriate antibiotics (section 4).

to:

  1. 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:

  1. For PstT1, incubate the KB plate at 28 ˚C for 48 h prior to using bacteria in the flood experiment.

to:

  1. 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:

  1. 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:

  1. 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:

  1. 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:

  1. 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:

  1. 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:

  1. 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:

  1. 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:

  1. 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:

  1. 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 ATissue Weight (g) Column B# of Colonies in a spot Column CDilution factor for spot2 Column DAdjusted # of Colonies3 Column EDilution factor for serial dilution Column FTotal # of Colonies Column G (cfu/0.01 g)4Average # of Colonies (cfu/0.01 g) Column HAverage Log Growth (cfu/0.01 g (log10)) Column I
Sample 10.004 g10200calculated as: (C2 x 0.01 g) / B2 = 251000calculated as: (D2 x E2 x F2) = 5000000average for sample 1 through last sample: (ie. average G1:G3) = 7000000log of average ie. log(H2) = 6.85
Sample 20.003 g1520050100010000000
Sample 30.002 g62003010006000000
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:

  1. 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 ATissue Weight (g) Column B# of Colonies in a spot Column CDilution factor for spot2 Column DAdjusted # of Colonies3 Column EDilution factor for serial dilution Column FTotal # of Colonies Column G (cfu/0.01 g)4Average # of Colonies (cfu/0.01 g) Column HAverage Log Growth (cfu/0.1 g (log10)) Column I
Sample 10.04 g10200calculated as: (C2 x 0.1 g) / B2 = 251000calculated as: (D2 x E2 x F2) = 5000000average for sample 1 through last sample: (ie. average G1:G3) = 7000000log of average ie. log(H2) = 6.85
Sample 20.03 g1520050100010000000
Sample 30.02 g62003010006000000
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:

  1. Phenotype plants infected with PstT1 at 10–14 days after flood inoculation.

to:

  1. Phenotype plants infected with Pst19 at 10–14 days after flood inoculation.

Figure 4 in the Protocol section was updated from:

Tomato resistance-susceptibility diagram; PstDC3000, PstT1 assay; cultivars, mapping analysis.
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:

Cultivar resistance diagram; tomato genotypes PstDC3000, Pst19, resistance vs. susceptibility analysis.
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:

Plant resistance assay; petri dishes with seedlings; MgCl₂, PstT1 infection; comparative growth study.
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:

Plant-pathogen interaction in petri dishes; tomato seedlings, MgCl₂ treatment, pathogen resistance.
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

Bar chart of bacterial growth; cfu/0.01g (log 10) comparison in PstT1 infection study.
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.

x

Bar chart comparing bacterial cfu in tomato cultivars, highlighting resistance levels; Pst19 experiment.
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.

to:

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.

to:

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.

Etiketler

Domates Fidesi Sel DeneyiPseudomonas Syringae DirenciY ksek Kapasiteli TaramaYabani Domates AksesyonlarFide nok lasyon ProtokolBakteriyel B y me DeneyleriPto Prf Gen K mesiHastal k Direnci FenotiplemesiTohum Sterilizasyon Y ntemiB y me Odas Ko ullar

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