Plant Physiology, February 2008, Vol. 146, pp.703–715 Salicylic Acid and Systemic Acquired Resistance Play a Role in Attenuating Crown Gall Disease Caused by Agrobacterium tumefaciens. Ajith Anand, Srinivasa Rao Uppalapati, Choong-Min Ryu2, Stacy N. Allen, Li Kang, Yuhong Tang, and Kirankumar S. Mysore* Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 Plant Physiology, February 2008, Vol. 146, pp.703–715

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the role of SA-mediated plant defense responses against Agrobacterium the direct effects of SA on microbes SA Inhibits Agrobacterium Growth in Vitro and Affects virulence SA Application on Plants Transgenic Plants Expressing NahG Silencing of SA Biosynthetic and Signaling Genes

1 2 3 4 6 7 8 GUS histochemical staining transient transformation assays 1 fluorimetrical assay 1- SA Application Stable transformation assays leaf discs tumor assay 2 2 - Transgenic Plants Expressing NahG transient transformation assays 3 3- effect Ag. Growth media 4 4- effect Ag. virulence leaf discs tumor assay GFP fluorescence assay 6 4- bacterial attachment assay serial dilution plating 7 6- Affects Ag. Gene Expression NbNPR1 (nonexpresser of PR gene) NbSABP2 (SA-binding protein) SlICS 8 5- Signaling Genes in

. Zhu Y et al. Plant Physiol. 2003;132:494-505 ©2003 by American Society of Plant Biologists

If SA Application on Plants Decreases Agrobacterium Infection exogenously applying SA to N. benthamiana through soil drenching Leaves from the SA-treated and mock-treated plants were collected 7 d posttreatment and subjected to: stable and transient transformation assays

T-DNA Binary system

Histochemical spectrophotometrical fluorimetrical The GUS reporter system (GUS: beta-glucuronidase) is a reporter gene system Histochemical spectrophotometrical fluorimetrical The purpose of this technique is to analyze the activity of a promoter (in terms of expression of a gene under that promoter) The technique is based on beta-glucuronidase, this enzyme, can transform some specific colorless or non-fluorescent substrates, into coloured or fluorescent products.

Transient and stable transformation ? SA-treated GV2260 (carrying the binary vector pBISN1) Leaf disck Incubate on CIM stained with X-Gluc staining N.benthamiana fluorimetrical

Quantification of transient transformation in the SA-treated plants The direct effects of SA on Agrobacterium-mediated plant transformation Quantification of transient transformation in the SA-treated plants The inoculated leaves were stained with X-Gluc staining solution a significant reduction in GUS activity

a significant reduction in GUS activity GUS activity was measured in GV2260- infected leaf discs at 2, 5, and 10 dpi by recording the fluorescence of 4-methylumbelliferone a significant reduction in GUS activity

the free SA using a mass spectrometry system SA treatment of N. benthamiana plants result in increased SA levels.

We therefore conclude that SA Application on Plants Decreases Agrobacterium Infection We therefore conclude that SA application partially block sA grobacterium-mediated planttransformation.

Transgenic Plants Expressing NahG Are they Hyper susceptible to Agrobacterium Infection? transgenic plants expressing salicylatehydroxylase (NahG), which degrades SA to catechol NahG-overexpressing plants wild-type tomato plants . Detached leaves of wild-type tomato plants (‘Moneymaker’)and NahG-overexpressing plants were vacuum infiltrated with the disarmed strain A. tumefaciens GV2260 (carrying the binary vector pBISN1) at a low concentration (1 3 105 cfu). Three days postinfection the leaves were stained with X-Gluc for detecting GUS expression.

These results suggest that SA plays a role in protecting plants against Agrobacterium infection.

If SA Inhibits Agrobacterium Growth in Vitro and Affects Its Virulence? SA may be an important determinant of Agrobacterium pathogenicity the direct role of SA on bacterial growth and and virulence

If SA Inhibits Agrobacterium Growth in Vitro? SA was added into the culture media at physiologically relevant concentrations was monitored in both the minimal and rich media Agrobacterium growth inhibition was observed when SA was supplemented in the minimal media However, SA at low concentrations (5–15 mM) did not affect the growth of the bacteria in the rich media

Effect of SA on Agrobacterium growth in different media and various concentrations of SA (0-15 μm).

if SA directly affects Agrobacterium virulence? leaf disc infection assays with A. tumefaciens A348 treated with SA. Agrobacterium attenuates its capacity to incite tumors on leaf discs exogenous application of SA reduce the virulence of Agrobacterium.

If Agrobacterium Treated with SA Is Defective in Attaching to Plant Cells? Agrobacterium attachment assay with the disarmed strain A. tumefaciens KAt153 (carrying the binary vector pDSKGFPuv) that was mock or SA treated SA at 100 mM affected Agrobacterium attachment (left panel: GFP fluorescence; right panel: epifluorescence image). SA may affect the virulence by interfering with the attachment of Agrobacterium to plant cells.

If SA Affects Agrobacterium Gene Expression? custom-made whole-genome Affymetrix microarrays SA treatment significantly affected the expression of the Ti plasmid genes 36 of the 37 genes were induced by AS at 4 h and 103 of the 172 genes were induced by AS at 24 h, respectively

SA repressed the expression of the bacterial virulence (vir genes), the conjugal transfer (tra genes), and plasmid replication genes (repABC operon), Using real-time quantitative reverse transcription-PCR (qRT-PCR), we confirmed the differential expression of few selected genes these results suggest that SA has multiple effects on Agrobacterium resulting in reduced virulence.

Silencing of SA Biosynthetic and Signaling Genes in N Silencing of SA Biosynthetic and Signaling Genes in N. benthamiana Increases Susceptibility to Crown Gall Disease relatively larger tumors on the shoots of ICS-, NPR1-,and SABP2-silenced plants compared with the tumors on Tobacco rattle virus (TRV)TGFP-inoculated and wild typ plants

Virus-Induced Gene Silencing (VIGS) Tobacco Rattle Virus (TRV) used as the VIGS vector RISC RISC AAAAAA RdRp- We insert a sequence of rna corresponding to the gene we want to silence into the TRV genome. RdRp = RNA dependent RNA polymerase Dicer RISC RNA induced silencing complex Plant cell

These results indicate that SA biosynthetic and signaling genes also play a significant role in antagonizing Agrobacterium infection..

BTH-Induced SAR Impairs Agrobacterium Infectivity In planta tumor assay smaller tumors were observed on shoots of BTH-treated N. benthamiana and tomato plants

BTH-Induced SAR Impairs Agrobacterium Infectivity Stable transformation assays Tumors produced on leaf discs derived from both silenced and wild-type plants were smaller in BTH treated plants

BTH-Induced SAR Impairs Agrobacterium Infectivity transient transformation Leaves treated with BTH showed a significant reduction in GUS activity at 2 and 5 dpi in both silenced and wild-type plants

both SA and SA-mediated plant defenses play a key role in determinin Agrobacterium infectivity on plants independent of SA, SAR is an important determinant in Agrobacterium infectivity both endogenous SA levels and SAR are critical determinants of Agrobacterium pathogenicity in plants

Besides triggering the defense responses, SA had direct effects on Agrobacterium fitness and virulence and therefore plays a central role in Agrobacterium-plant interactions. we speculate that SA competes with AS for direct or indirect interaction with VirA

exogenous application of SA or its analogs before the onset of crown gall disease presents a possible means for achieving durable disease control.