1. 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

2. What's your idea ?

3. 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

4. 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

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

6. 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

7. T-DNA Binary system

8. 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.

9. 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

10. 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

11. 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

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

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

14. 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 ( cfu). Three days postinfection the leaves were stained with X-Gluc for detecting GUS expression.

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

16. 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

17. 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

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

19. 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.

20. 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.

21. 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

22. 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.

23. 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

24. 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

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

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

27. 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

28. 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

29. 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

30. 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

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