Kevin Ann Hunt
Biol 509- Molecular Biology and Applications
1 December 2010
Séverine Lacombe, Alejandra Rougon-Cardoso, Emma Sherwood, Nemo Peeters,
Douglas Dahlbeck, H Peter van Esse, Matthew Smoker, Ghanasyam Rallapalli,
Bart P H J Thomma, Brian Staskawicz, Jonathan D G Jones & Cyril Zipfel.
“Interfamily transfer of a plant pattern-recognition receptor confers broadspectrum bacterial resistance.” Nature Biotechnology 28: 365 – 369. (April 2010)
Objective
Phytopathogenic bacteria heavily impact food production and agriculture by causing
massive losses. There are multiple ways to inhibit virulent bacteria in crops. The current
leading manner of inhibition is agrochemical applications; however, this treatment has
associated financial, health, and environmental costs. Increasing the plant’s innate
immune response could provide a more sustainable method of treatment. Plants protect
themselves with constitutive physical and chemical barriers and actively recognize
microbial invaders. Detection of potential pathogens is completed via two triggered
immunity systems: effector-triggered immunity (ETI) and PAMP-triggered immunity
(PTI). ETI is caused by intracellular resistance proteins (R proteins) that recognize
microbial virulence effectors. This type of immunity is gene-for-gene resistance; a plant
species may contain an R gene that matches a virulence effector carried by a limited
number of pathogenic microbes. Pathogens often evolve to overcome this type of
narrow resistance. PTI is carried out by Pattern-recognition receptors (PRRs) that detect
conserved microbial molecules, called pathogen-associated molecular patterns (PAMPs).
Unlike the virulence effectors of ETI, PAMPs are often essential and highly conserved so
evolution to avoid resistance is less likely. This article focuses on the Arabidopsis
thaliana PRR EFR. EFR recognizes the PAMP elongation factor Tu (EF-Tu); this PAMP is
highly conserved across bacteria. Thus, the interfamily transfer of EFR offers the
possibility of broad-spectrum resistance to bacterial pathogens.
Experiments and Results
To confirm that EFR detects EF-Tu proteins from important plant virulent bacteria, first
the variability and eliciting activity of elf18 peptides (EF-Tu eliciting epitope) from
phytopathogenic bacteria of several genera was assessed in A. thaliana. An alignment
of 19 elf18 sequences allow for their division into 7 groups A-G (Fig. 1a). A
representative of each of the 7 groups was used to measure eliciting activity in A.
thaliana leaves; each induces an oxidative burst (Fig. 1c) indicating the various elf18
sequences elicit a response. Heat-killed bacterial soluble extracts of three
phytopathogenic bacteria was used to treat wild-type, efr knock-out, fls2 knock-out,
and fls2/efr knock-out A. thaliana. FLS2 is a PRR that recognizes the PAMP flagellin. A
response to the extracts, which contained EF-Tu, elicited activity (oxidative burst) in the
wild-type and the fls2 stains, but not the strains with EFR removed (Fig. 1d). This
indicates that the major PAMP in these extracts that is recognized by A. thaliana is EFTu. All of the phytopathogenic bacteria tested demonstrated EF-Tu eliciting activities,
confirming the possibility of broad-spectrum resistance.
The next step was to transform two solanaceous species with EFR and determine their
responsiveness to EF-Tu. Wild-type and EFR-expressing Nicotiana benthamiana and
Solanum lycopersicum were treated with elf18 or flg22; the EFR-expressing plants
produced a response to elf18 where the wild-type did not (Fig. 2b,e). RtPCR was done
for wild-type and EFR-expressing N. benthamiana after treatment with flg22 or elf18 for
0, 30, 60, and 180 minutes. Treatment with elf18 caused an increase in expression of
defense-marker genes in EFR-expressing plants solely (Fig. 2c). Wild-type and EFRexpressing N. Benthamiana seedlings were treated with elf18 or flg22. The growth of
EFR seedlings was inhibited by the presence of elf18 (Supp. Fig. 1). The introduction of
a new receptor may affect the function of preexisting receptors. The purpose of using
flg22, a peptide of the most conserved domain of flagellin, in the above experiments
was to determine if the presence of EFR interfered with the normal action of FLS2. EFR
did not cause a reduction in the response elicited by flg22 in N. benthamiana or S.
lycopersicum (Fig. 2b,e). RtPCR revealed flg22 caused expression of similar amounts of
defense-marker genes in both wild-type and EFR-expressing N. Benthamiana (Fig. 2c).
Treatment of seedlings with flg22 caused a reduction in growth in both wild-type and
EFR-expressing N. Benthamiana (Supp. Fig. 1). Thus, the transfer of EFR has no
detrimental effects on the endogenous levels of FLS2. Another potential problem
associated with the introduction of defense-related genes is constitutively activated
defense responses which can cause necrosis and/or reduced growth. RtPCR was
completed for defense marker genes in wild-type and transgenic EFR N. Benthamiana;
EFR did not cause any constitutive increase in defense gene production (Fig. 2c; Supp.
Fig. 4). To further indicate this, wild-type and transgenic EFR N. Benthamiana and S.
lycopersicum were treated with Veticillium dahlia, a fungal pathogen. Transgenic EFR
plants were not more resistant than the wild-type plants (Supp. Fig. 5).
Next, the ability of EFR to confer disease resistance to adapted virulent bacteria was
tested. N. Benthamiana strains were treated with Pss B728a, the causal agent of
bacterial brown spot in bean, or Pta 11528, the causal agent of tobacco wildfire. In
both treatments EFR conferred resistance to the P. syringae pathovars, reducing growth
on leaves to 1.25% and 0.02%, respectively, of wild-type growth 4 days past
inoculation (Fig. 3a,b). N. Benthamiana strains were also treated with an Agrobacterium
tumefaciens strain causing transient expression of GUS and an A. tumefaciens virulent
tumorigenic strain. EFR impaired the transient expression of GUS (Supp. Fig. 2) and
decreased tumors by ~75% (Fig. 3c). Solanum lycopersicum wild-type and transgenic
EFR strains were treated with major pathogens, either the vascular phytopathogenic
bacteria Ralstonia solanacearum, the causal agent of bacterial wilt disease, or the foliar
phytopathogenic bacteria Xanthomonas perforans, the causal agent of bacterial spot
disease. EFR drastically reduced the effect of bacterial wilt disease (Fig. 4a,b) and
conferred resistance to X. perforans, although to a lesser extent than other bacteria.
EFR was less effective in reducing X. perforans than the Bs2 gene from pepper (Fig. 4c;
Supp. Fig. 3). The Bs2 gene encodes an R protein specific for an effector conserved in
X. perforans.
Conclusion
The transfer of PRRs across plant families provides several advantages over current
bacterial treatments. A reduction in agrochemical inputs is possible. Transgenic
approaches can be done quickly and in crops where classical breeding is difficult. PRRs
have the potential to confer broad-spectrum resistance. PAMPs are often highly
conserved and essential, thus it is unlikely for a pathogen to develop resistance,
because mutations in PAMPs might reduce fitness. Also, the huge reduction in bacterial
growth on the plants in these experiments decreases the probability for the breakdown
of resistance. However, eventual recognition escape or PTI suppression cannot be
discounted. This work provides a novel biotechnological approach for disease
resistance. The authors indicate that a combination of PRRs or PRRs and R proteins
could be used to create resistance to a wide range of plant pathogens with a high
potential for durability.