Xinnian Dong, HHMI Investigator, Arts & Sciences Professor of Biology, Duke University • Expression of broad-spectrum disease resistance in plants often results in fitness penalty. • Plant immune responses are normally regulated at both transcriptional and translational levels. • Transcriptional and translational regulatory mechanisms of plant immune genes can be manipulated to engineer broad-spectrum resistance in crops.

1. Precision in plant immune expression:
Not lost in translation
Xinnian Dong

2. NPR1 is a master immune regulator required
for SA-mediated gene expression
Cao et al. (1994) Plant Cell 6:1583
Wang et al., (2006) PloS Pathogens e123

3. A partial list of NPR1 target genes
Wang et al. Science 2005

4. TL1 is enriched in SA-induced ER gene promoters
Wang et al. Science 2005

5. Identification of TBF1 (HSF4) as a TF for TL1
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 - 112

6. TBF1 activates defense but inhibits growth genes
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 - 112

7. Canet et al., (2010) Plant, Cell & Environment 33:1911
Constitutive induction of immunity
leads to inhibition in plant growth
npr1

8. TBF1 regulates growth-to-defense transition
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 - 112

9. TBF1 transcription is SA-inducible
in an NPR1-dependent manner
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 - 112

10. TBF1 translation is regulated by two uORFs
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 - 112

11. Translation in eukaryotes

12. Translation in eukaryotes

13. Translation in eukaryotes

14. Translation in eukaryotes

15. Translation in eukaryotes

16. Translational repression of uORFs
is alleviated upon elf18 treatment
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 – 112
Xu, Greene, Yoo et al., 2017, Nature 545:487

17. Translation derepression through uORFs
is a common mechanism for triggering immunity

18. TBF1 translation is proposed to be activated
upon pathogen-induced perturbation in Phe
Pajerowska-Mukhtar et al. (2012) Curr. Biol. 22:103 – 112

19. GCN2-mediated phosphorylation of eIF2a is
not required for translation during PTI or ETI
Xu, Greene, Yoo et al., 2017, Nature 545:487

20. uORFs are prevalent in eukaryotic mRNAs
& have signal-specific effects on translation
Yeast: 13% of all mRNAs (Lawless et al. BMC Genomics
10:7).
Arabidopsis: 5360 potentially translated uORFs in 2051
genes. GO terms enriched for proteins with catalytic,
binding, transferase, phosphotransferase, kinase, and
transcription regulator activities (Hu et al., IEEE
TRANSACTIONS ON NANOBIOSCIENCE 15:148).
Humans: 49% of all mRNA including two-thirds of
oncogene mRNAs and transcripts that encode proteins
involved in important cellular processes, such as
differentiation, cell cycle, and stress response (Barbosa
et al., 2013, PloS Genetics 9:e1003529).

21. Pathogen X
Pathogen Y
Pathogen Z
Current Technology
Introducing one R-gene
at a time via traditional
plant breeding,
transgenic or CRISPR
technology. Pathogen
effector specific and
may not be effective
against fast evolving
pathogens.
Pathogen X
Pathogen Y
Pathogen Z
Current Technology
Expressing broad-
spectrum disease
resistance without
translational control
often has yield
penalties.
Pathogen X
Pathogen Y
Pathogen Z
Our Technology
Transcriptional &
translational control
of broad-spectrum
resistance avoids yield
penalties and reduces
selection pressure on
resistant pathogens.
Adding translational control to
engineering disease resistance in crops

22. uORFTBF1 is stringent enough
to inhibit TBF1-mediated PCD
Xu and Yuan et al. 2017, Nature 545:491

23. uORFTBF1-mediated translational induction
occurs rapidly and transiently
Xu, Greene, Yoo et al., 2017, Nature 545:487

24. uORF2TBF1 sequences (DNA & protein)
are evolutionarily conserved in plants
Xu and Yuan et al. 2017, Nature 545:491

25. Engineering broad-spectrum resistant rice
through TBF1-controlled production of AtNPR1
Xu and Yuan et al. 2017, Nature 545:491
TBF1p: uORFsTBF1 -NPR1
The TBF1 promoter
(TBF1p) is rapidly
induced upon pathogen
challenge.
The uORFs in the TBF1
leader sequence block
spurious translation w/o
pathogen challenge. This
inhibition is immediately
alleviated upon pathogen
challenge.
NPR1 is a master immune
regulator whose over-
expression has been
shown to confer broad-
spectrum resistance in
multiple crops plants
including rice and wheat.

26. The TBF1p:uORFTBF1-NPR1 transgenic rice
shows normal growth & seed yield
Xu and Yuan et al. 2017, Nature 545:491

27. Engineering broad-spectrum resistant rice
through TBF1-controlled production of AtNPR1
Xu and Yuan et al. 2017, Nature 545:491

28. Pathogen stress can lead to
large yield losses in rice
2017/2018 global rice production estimated at 483 million metric tons.
Pathogens estimated to decrease overall yields between 24-41%.
Estimated cost of yield loss up to $74B.
Cost of mitigating pathogen outbreaks via fungicides is prohibitive in
many countries.
Fungus
Bacteria
Viruses

29. By Meng Yuan
Major rice diseases
(*known to be controlled by NPR1 overexpression)
Ÿ Rice blast*
Ÿ Rice sheath blight*
Ÿ Rice false smut
Ÿ Rice bacterial leaf blight*
Ÿ Rice bacterial leaf streak*
Ÿ Rice virus diseaseYield loss

30. Summary
(1) Translation is an important layer of regulation during immune
induction with most of the players and mechanisms still unknown.
(2) During PTI induction, translational induction occurs rapidly and
transiently prior to transcriptional activation.
(3) The two uORF in the TBF1 mRNA tightly inhibit its basal
translation and this inhibition is rapidly alleviated through an
unknown mechanism upon pathogen challenge.
(4) The TBF1-cassette (promoter & leader sequence) can be used to
produce defense proteins (snc1 & NPR1) to confer broad-spectrum
resistance without compromising plant fitness.

31. Acknowledgements
Karolina Pajerowska-Mukhtar, Wei Wang, Yasuomi Tada,
Nodoka Oka, Chandra Tucker, Jose Pedro Fonseca
Guoyong Xu, George Greene, Heejin Yoo, Lijing Liu,
Jorge Marques, Jonathan Motley
Guoyong Xu, Meng Yuan, Chaoren Ai, Lijing Liu, Edward
Zhuang, Sargis Karapetyan, Shiping Wang