1. By Sherry S. and Sarzana H.
Isochorismate synthase is required to synthesize salicylic acid for plant defence
Mary C. Wildermuth, Julia Dewdney, Gang Wu & Frederick M. Ausubel
NATURE, 2001
By Sherry S. and Sarzana H.

2. Recap on Plant Defence and SAR
Systemic Acquired Resistance (SAR) = whole-plant resistance response to pathogen exposure
Localized exposure of pathogen triggers resistance in distil parts of the plant
(SAR) is a "whole-plant" resistance response that occurs following an earlier localized exposure to a pathogen. SAR is analogous to the innate immune system found in animals, and there is evidence that SAR in plants and innate immunity in animals may be evolutionarily conserved.[1]

3. Significance of Salicylic Acid (SA) in SAR
SA level are increased locally and systemically upon SAR induction
SA is required for SAR SA

4. SA Biosynthesis: the Shikimic Acid Pathway
The shikimate pathway (shikimic acid pathway) is a seven step metabolic route used by bacteria, fungi, algae, some protozoan parasites and plants for the biosynthesis of folates, carbs, and aromatic amino acids (phenylalanine, tyrosine, and tryptophan).
The plant shikimate pathway is the entry to the biosynthesis of phenylpropanoids (C6–C3) and subsequent natural products including lignans and stilbenes

5. SA Biosynthesis in plants
In 2001, SA believed to be critical for HR response
SA synthesized from Phenylalanine pathway
How can plants still make SA when Phenylalanine pathway knocked down? ?

6. SA Biosynthesis in bacteria
Pseudomonas aeruginosa possess isochorismate synthase (PchA)
Chorismate → Isochorismate
Do plants have their own ICS?
pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively
Mauch et al. 2001

7. SA Biosynthesis in plants
Pseudomonas aeruginosa and other bacteria possess isochorismate synthase (ICS)
Chorismate → Isochorismate
Do plants have their own ICS?

8. SA Biosynthesis in plants
Basal SA levels
SA Biosynthesis in plants
Huang et al. (2010) made quadruple mutant pal1-4
total SA levels were about 50% of wild-type levels after the pathogen infection in pal1-4
Induced SA levels

9. Some Big-Picture Questions:
Does Arabidopsis putative ICS protein synthesize SA?
Which gene encodes ICS1 ?
Does ICS1 have a role in SAR?
How is ICS1 regulated?
Where does Phenylalanine-mediated SA synthesis fit in?

10. Connection between ICS, SA, and SAR? SA
SAR
In addition, when Arabidopsis plants were inoculated with an avirulent strain of the Pseudomonas pathovar maculicola, resulting in SA accumulation and SAR in systemic leaves, ICS1 was systemically induced (data not shown).

11. ICS1 co-expression with PR1
A. thaliana infected with virulent Erysiphe orontii and avirulent bacteria Pseudomonas syringae pv. Maculicola, ICS1 expression induced along with PR1
White = PR1
Black = ICS1
To test the hypothesis that plants use ICS to synthesize SA which os used in SAR, we first examined whether pathogen treatment of Arabidopsis induces either of these ICS genes. Expression of ICS1 occurred in leaves infected with the fungal biotroph Erysiphe orontii or the bacterial necrotroph Pseudomonas syringae pv. maculicola.

12. ICS1 induced systemically
Inoculation with Pseudomonas pv. maculicola leads to SA accumulation + SAR
ICS1 induced systemically
White = PR1
Black = ICS1
To test the hypothesis that plants use ICS to synthesize SA which os used in SAR, we first examined whether pathogen treatment of Arabidopsis induces either of these ICS genes. Expression of ICS1 occurred in leaves infected with the fungal biotroph Erysiphe orontii or the bacterial necrotroph Pseudomonas syringae pv. maculicola.

13. Connecting ICS1 to sid2 mutants
What phenotypes should ics1 mutant have?
low/no SA accumulation
Decrease in expression levels of pathogenesis-related genes (PR1, etc.)
Enhanced disease susceptibility
Implications that the ICS1 protein is encoded by sid2 gene:
a. They hypothesize that an ics1 mutant would have the following phenotypes:
i. Low/no SA accumulating
ii. Decreased levels of pathogenensis-related genes expressed
iii. Enhanced disease susceptibility

14. Connecting ICS1 to sid2 mutants
Previous studies show sid2 mutants have
Low SA accumulation
Decrease in PR gene expression levels (PR1)
Enhanced susceptibility to disease
sid2-1
Nawrath and Métraux 1999
sid2-2
Implications that the ICS1 protein is encoded by sid2 gene:
a. They hypothesize that an ics1 mutant would have the following phenotypes:
i. Low/no SA accumulating
ii. Decreased levels of pathogenensis-related genes expressed
iii. Enhanced disease susceptibility
Dewdney et al. 2000

15. sid2-2 Fast neutron mutagenesis (deletions)
Northern Blot Analysis
Fast neutron mutagenesis (deletions)
No ICS1 transcript detected post infection
Implications that the ICS1 protein is encoded by sid2 gene:
b. Previous studies show that sid2 mutants have all these phenotypes
i. And sid2 gene has been mapped to bottom of bottom of chromosome 1 (near ICS1 locus)

16. sid2-1 and sid2-2 mutants sid2-1 and sid2-2 are allelic
Mapped to bottom of chromosome 1, near ICS1 locus
sid2-1: point mutation in exon 9
Implications that the ICS1 protein is encoded by sid2 gene:
b. Previous studies show that sid2 mutants have all these phenotypes
i. And sid2 gene has been mapped to bottom of bottom of chromosome 1 (near ICS1 locus)

17. sid2-1 and sid2-2 mutants
sid2-2: at least 50bp deletion in exon 9 (fast neutron mutagenesis)
ICS1 primers for exon 9 region shows abnormal PCR product (Southern Blot, fig. b).
Implications that the ICS1 protein is encoded by sid2 gene:
b. Previous studies show that sid2 mutants have all these phenotypes
i. And sid2 gene has been mapped to bottom of bottom of chromosome 1 (near ICS1 locus)

18. What is the structural organization of the Arabidopsis ICS1 protein?
Now that we have looked into

19. Arabidopsis ICS1 protein: N-terminal
Compared Arabidopsis ICS1 mRNA sequence w/ those of other plants
Arabidopsis ICS1 had extra N-terminal
Sequenced the cDNA from ICS1 mRNA – via RACE PCR
Technique allowing for amplification of mRNA ends
Found a 66 AA N-terminal (marked by green)
Sequence represents a putative chloroplast transit signal
Also has a cleavage site (putative)

20. Rapid amplification of cDNA ends – PCR (RACE-PCR)
RT-PCR
RACE-PCR
Add a phosphotase - which dephosphorylates any uncapped or degraded (non-full length) mRNA
Then you add an enzyme to get rid of the 5’ methylated cap
This exposes the phosphate the 5’ end
3. Now its time to add an RNA oligotide linker that’s intended to attach on to the 5’ end (with the help of a ligase)
The sequence of this linker is known
4. Primers specific to the gene will bind somwehre along the mRNA - Reverse Transcriptase will now make the complimentary DNA stransd (we have our first DNA strand)
4. Then DNA primers representing the linker sequence bind to the 3’ end of this cDNA - giving us the second cDNA strand synthesized up to the region of where the gene specific primer is
Yeku and Frohman, 2010
RACE-PCR

21. Arabidopsis ICS genes possibly acquired from prokaryotes
Endosymbiotic theory
Plastid organelle derived from cyanobacterium endosymbiont
Hypothesized that many genes from the plastid ancestor transferred to the nucleus
Partial conservation of PHYLLO locus across plants, algae, and bacteria
PHYLLO locus = construct of 4 men genes
menF = strong homology to plant ICS1 & ICS2
Support for plant ICS genes being of bacterial origin
The Phyllos locus has been studied for investigating vitimin K biosynthesis
menF gene is found in plastomes (plastid genome) of the red algae
Trichodemium erythraeum is a cyanobacteria
Gross et al., 2006
Raven and Allen, 2003

22. Arabidopsis ICS1 protein: C-terminal
Carboxyl end = highly conserved chorismate-binding domain
Homology with known ICS proteins in other bacterial & plant species
Alignment of chorismate-binding domains across of 5 proteins
Comparison of complete AA sequence of Arabidopsis ICS to other ICS
This domain = about 200 amino acids long
Blue asterisk = residues deemed vital for ICS activity
Black boxes = identical across the different
Grey boxes = conserved AA in some proteins

23. ICS proteins of Catharanthus roseus confirmed to make Isochorismate
Purified ICS from Madagascar periwinkle - showed in vitro acvitity of converting chorismate to isochorismate => and followed typical Michaelis-Menton enzyme kinetics
Has over 50% AA similarity to Arabidopsis ICS1 = good sign for actual biochemical acitivty of A. thaliana ICS1

24. Where does ICS fit in the model for SAR response?

25. Evidence supporting SA produced via ICS1 is necessary for SAR
nahG mutants = deficient SAR upon pathogen infection
Decreased levels of SA
sid2 mutants:
Reduced ICS1 expression
SA levels 5-10% that of WT
PR-1 gene expression 1-10% that of WT
17 dpi with E. orintii
Systemic leaves show susceptibility to infection after local leaves inoculated w/ fungal pathogen (previous studies)
Reduced ICS1 expression
Reduced SA levels
Reduced PR-1 expression
RNA of ICS and PR1 detected via Norther blot
Susceptibility of systemic leaves to fungal pathogen infection (whereas WT is resistant to its growth on systemic leaves) - published in Dewdney et al. ‘s previous paper (2000)
/sid2
Dewdney et al., 2000

26. Evidence supporting SA produced via ICS1 is necessary for SAR
cpr mutants
Constitutively PR-1 expression and SA levels
Constitutively active SAR
Check for ICS1 expression in cpr mutants
Positive correlation: Constitutive SAR coincides with constitutive ICS1 expression + high SA levels
Constitutive expression of ICS1
Legend
= 1/10 of WT levels
= 1/2 of WT levels
WT = WT level of expression
+ = 2x WT levels
++ = 10x WT levels
Constitutive (and elevated levels) of ICS1 expression
Correlation between constitutive SAR induction and constitutive ICS1 expression

27. Where does ICS1 act relative to NPR1?
npr1 mutants = deficient SAR response to pathogen infection
NPR1 found to work downstream of SA
Works in concert with TFs to regulate PR gene expression
Measure ICS1 expression in npr1 background:
Legend
= 1/10 of WT levels
= 1/2 of WT levels
WT = WT level of expression
+ = 2x WT levels
++ = 10x WT levels
ICS1 is likely functioning upstream of NPR1
Ask audience what this suggests - about NPR1 activity

28. Is NPR1 a negative regulator of ICS1 expression?
Results support NPR1 negatively regulating ICS1 expression
If NPR1 was a negative regulator, in the absence of NPR1 we expect:
Higher levels of ICS1 expression and consequently SA levels upon infection of plant
ICS1 expression and SA levels relative to WT in npr1 mutant:
Legend
= 1/10 of WT levels
= 1/2 of WT levels
WT = WT level of expression
+ = 2x WT levels
++ = 10x WT levels
Supports hypothesis that the NPR1 acting downstream - has a repressive regulatory function on ICS1 upstream

29. Does SA autoregulate expression of ICS1?
Hypothesis: SA affects the gene expression of ICS1
To test this hypothesis:
Measured ICS1 expression in the nahG mutant background
SA alone does not induce or repress ICS1 expression
Legend
= 1/10 of WT levels
= 1/2 of WT levels
WT = WT level of expression
+ = 2x WT levels
++ = 10x WT levels
Since nahG has diminished levels of SA and compromised SAR (as noted by much lower expression of PR1 genes)
Absence of SA - does not change ICS1 expression in nahG plants
Ask audience what this suggests about SA regulating ICS1 expression
They conclude that SA alone is not sufficient to impact/regulate ICS1 expression

30. Predicted Chain of Action for SAR Response
In absence of functional ICS1 - do not get PR1 expression or elevated levels post pathogen infection
We know NPR1 is downstream ICS1 activity
NPR1 is likely a negative regulator of ICS1 (come work upstream)
SA likely does not go back and affect ICS1 expression in an autoregulatory
In constitutively active SAR mutants - ICS1 is also consitutively turned on

31. How is ICS1 expression regulated?
Clueing into the ICS1 promoter region
Check promoter sequence for cis-regulating elements
Found many W-box elements
W-box elements = sequence recognized by WRKY (TF)
Found in PR1 promoter
Also in promoter of CM1 (encodes chorismate)
= W-box elements
CM1 protein encodes a protein that bind chorismate (substrate)

32. Role of WRKY in Response Regulation
Family of plant-specific TF
Involved in BOTH abiotic and biotic stress responses
Evidence for different WRKY factors acting as + or - regulators
WRKY domain + C/H residues (making up the zinc finger-like motif) = high affinity binding to W-box elements
Bakshi and Oelmuller, 2014

33. Role of WRKY in Response Regulation
A general diagram showing the network of signal transductioin induced by abiotic or biotic stressors
There’s Calmodulin and MAPKinases using phosphorylatio activation to pass on signal
This results in promoting expression of WRKY genes or activation of their function (phosphorylating = activation)
Some WRKYs behave as a repressor of physiological processes (marked by X)
Bakshi and Oelmuller, 2014

34. Hypotheses of how WRKY regulates genes in SAR response
Different WRKY factors act on ICS1 and downstream genes
Early and late-acting WRKY factors activated by SA, pathogens, and other elicitors regulate ICS1 and PR1
Shared WRKY factors may act on both ICS1 and PR1
One potential explanation consistent with an enrichment of W-box elements in both the promoters of ICS1 and downstream genes (for example, PR1) is that different WRKY factors regulate these responses. Reports of a variety of early- and late-acting WRKY factors activated by SA, elicitors and/or pathogens25 support this view. Alternatively, shared WRKY factor(s) may suppress both ICS1 and PR1 expression.

35. Hypotheses of how WRKY regulates genes in SAR response
LS4 = a negatively regulated domain in a W-box element
LS7 =bZIP transcription factor binding domain

36. How is ICS1 expression regulated?
Clueing into the ICS1 promoter region
ICS1 promoter has Myb-binding domains
Also found on CM1 and ASA1 (proteins which also bind chorismate)
Myb = TFs that help regulate
pathogen response
2 metabolism
ASA1
CM1
MYB factors - unlike WRKYs - is found in all eukaryotes
MYB factors are represented in all eukaryotes - contribute to a whole variety of functions
Organized into classes based on the number of MYB repeats they have in the MYB DNA-binding domain
Plants most abundantly have the R2R3 class of MYB factors - responsible for all these processes including responses to pathogen threats
Also mentioned that ASA1 and CM1 have these MYB-binding domains (not on PR1)
Unique to chorismate using enzymes
ASA1 and CM1 go involved in the immune response needed to make antimicrobials such as phytoalexins + alkaloid compounds
Hu et al., 2009
Ambawat et al., 2013

37. PAL possibly makes background SA
At the time, thought SA required for cell death
sid2 mutants still show cell death, and sid2 are a large contributor of SA
Perhaps PAL pathway produces SA facilitating necrosis?

38. Alternatively ICS2 gene possibly involved in low constitutive levels of SA
ICS2 could express constitutively low levels of SA
In sid2 mutants
In parallel - we see low constitutive levels of anthranilate in ASA1 mutants
Attributed to ASA2 protein

39. Conclusions ICS1 shows levels of expression upon pathogen infection
During SAR induction
sid2 background - see mutations in ICS1 gene
Consistent with the mutagenesis to obtain sid2 mutants
Support for sid2 gene encoding ICS1
SA produced by ICS1 - needed for SAR
Sid2 mutants = PR1 expression & SA levels & ICS1 expression after infection w/ pathogen
Constitutively active SAR = constitutively expressed ICS

40. Conclusions N-terminal = putative chloroplast transit sequence
ICS genes speculated to be of prokaryotic ancestry
C-terminal = conserved chorismate-binding domain
Promoter region has
W-box elements
MYB-binding sequence
PAL produced Isochorismate likely has a lower contribution to SA biosynthesis than the ICS genes
ICS2 predicted to produce low levels of SA in absence of ICS1 activity (sid2 mutants)