How has Lr34/Yr18 conferred effective rust resistance in wheat for so long?

BGRI Technical Workshop 2012, Beijing, 2.9.2012 Beat Keller

Institute of Plant Biology, University of Zurich, Switzerland

The Holy Grail of resistance breeding: Durable resistance

Resistance is defined as „durable“ when it remains effective in cultivars that are widely grown for long periods and in environments favorable to the disease (Johnson 1983). There are single, major resistance genes which are durable, but these are exceptions Durable resistance is mostly polygenic and caused by genes acting quantitatively

Lr34: a durable leaf rust resistance gene

R S

Thatcher + Lr34 Thatcher

•  Has been effective against leaft and stripe rust for more than 100 years in the field, certainly 40 years at large scale

•  Confers partial resistance

•  Prolongs the latency period and reduces the production of spores (slow-rusting gene)

•  Is associated with the morphological trait Leaf Tip Necrosis (Ltn1)

•  Is not race specific

Lr34 is a durable disease resistance gene

Lr34 confers resistance against multiple pathogens

Pm38

Bdv1

Lr34

Yr18

All these effects are caused by a single gene

cv.‘Arina‘: Swiss winter wheat, susceptible cv.‘Forno‘: Swiss winter wheat, durably resistant

Durable leaf rust resistance in the Swiss cultivar ‘Forno’

The Lr34 gene is also an important determinant of durable resistance in winter wheat, i.e. it is active in a broad germplasm and highly differing climatic conditions

Documented history of Lr34 begins at the end of the 19th century in Italy and is restricted to hexaploid wheat

Adapted from Kolmer et al. 2008. Crop Science. Pedigree of Lr34/Yr18 cultivar entries in different wheat breeding programs.

Italy

China

Marco Polo?

Lr34 is present in many cultivars released since the 1970ies, but not in older cultivars McCallum et al. Euphytica, 2012

Deployment of Lr34: the example of Canadian germplasm

Proportion of the total Canada Western Red Spring seeded area which was seeded to Lr34 carrying cultivars. Data from Canadian Wheat Board varietal surveys (McCallum and DePauw 2008). No prairie wide variety surveys were conducted 1993–1997

Eight open reading frames within a 363 kb target interval

Lr34 7DS 7DL Wheat chrom. 7D

Physical map ‘Chinese Spring’ (363 kb)

Hexose carrier

ABC transporter

Cytochrome P450

Cytochrome P450

Lectin receptor kinase

Cysteine proteinase (Ψ)

Glycosyl transferase (Ψ)

120,000 363,640

Genetic map chromosome 7DS

Xgwm1220 XSWSNP3 XcsLVE17 XSWM10

0.75 0.12

XSWSNP2 XSWDEL1 XSWDEL2 XSWDEL3

Lr34

0.03 0.03

SWDEL1 SWDEL2 SWDEL3 SWSNP2

Map-based cloning of the Lr34 gene

Hexose carrier

ABC transporter

Cytochrome P450

Cytochrome P450

Lectin receptor kinase

Cysteine proteinase (Ψ)

Glycosyl transferase (Ψ)

120,000 363,640

1 kb

ATG TAA

4E 3E m19 2G 4C 2F m21 2B

Eight Lr34 mutants: for example splice site mutations

Infection experiments revealed that the mutants are more susceptible to leaf rust, stripe rust, powdery mildew and stem rust and lost leaf tip necrosis

The molecular basis of the resistance effect of the Lr34 gene: Lr34 encodes a putative ABC transporter (ABCG or PDR) protein

What about Lr34-type of gene in susceptible lines (orthologous region in susceptible lines)? àSequence analysis in reference wheat lines with and without Lr34 Findings: Lr34- lines also contain the ABC transporter coding gene (allele Lr34sus) There are only three sequence differences between Lr34res and Lr34sus lines. Because of the dominant nature of Lr34res, these are gain-of-function mutations

1 kb

ATG TAA

Lr34res: 5’-CCGACTT-3’ Lr34sus: 5’-CCGTCTT-3’

Lr34res: 5’-TCC ATC ATG-3’ Lr34sus: 5’-TCC ATC TTC ATG-3’

Lr34res: 5’-TCG CAG CAT CGA-3’ Lr34sus: 5’-TCG CAG TAT CGA-3’

Deletion of a phenylalanine residue in ‘Chinese Spring’

Conversion of tyrosine to histidine in ‘Chinese Spring’

Resistant and susceptible allele differ by only two amino acid polymorphisms

Sequence differences between susceptible and resistant Lr34 alleles

Development and application of molecular markers for the Lr34 gene: • Perfect markers derived from the gene sequence

• Markers have been adopted in the last two years in most wheat breeding programs worldwide where leaf and/or stripe rust is of relevance

Functional studies of the Lr34 gene in transgenic wheat: cold-treated seedlings

Seedlings of transformed Bobwhite Cold treatment at 4oC

Risk, Selter et al. 2012 Plant Biotech. J.

Functional studies of the Lr34 gene in transgenic wheat: flag leaf of adult plant

Risk, Selter et al. 2012 Plant Biotech. J.

Microscopic analysis of leaf rust infection using WGA-alexa staining

Risk, Selter et al. 2012 Plant Biotech. J.

Risk, Selter et al. 2012 Plant Biotech. J.

Leaf tip necrosis is identical in Lr34 transgenic wheat

Genetic background of transgenic wheat plays an important role: seedling resistance in BW26SUI but not BW26AUS at 20oC

Risk, Selter et al. 2012 Plant Biotech. J.

à  Additive interaction(s) with other gene(s)  

Conclusions from transgenic wheat lines with Lr34

•  The transgene is fully functional against leaf rust and results in LTN

•  This complementation demonstrates that Lr34 is indeed pleiotropic

•  The genetic background can result in improved resistance: one should broadly cross the most active transgene into a broad set of breeding material (…normal breeding procedure…)

•  Combination with other durable resistance genes in a cassette or by crossing seems a good strategy

Why durable, how does the gene confer resistance? Some evolutionary findings and conclusions

PDRs in Arabidopsis and rice

There are 15 PDRs in Arabidopsis and 23 PDRs in rice

There is one clear Lr34 homolog in rice, but none in barley, Brachypodium and Arabidopsis

Closest homolog of Lr34 in rice: PDR23 (86% identity at AS level) second hit: OsPDR2 (55% identity) in Arabidopsis : PDR5 / PDR9 (56% identity at AS level)

Observations from the molecular nature of LR34: • It is not an NBS-LRR encoding protein (such as e.g. Lr1, Lr10, Lr21, the other cloned Lr resistance genes)

• This suggests a completely different molecular mechanism of resistance

• For example by reducing the quality of the leaves as «food» for biotrophic pathogens

• Transporting an antimicrobial substance (phytoalexins)

• Priming of resistance response?

Lr34 encodes an ATP-binding cassette (ABC) transporter

Transporters sharing a common basic structure: Nucleotide binding fold (NBF) Trans-membrane domain (TMB) Lr34 belongs to the subfamily of the pleiotropic drug resistance (PDR) transporters PDRs are only found in plants and fungi.

Lr34 protein: 1402 amino acids

NBF

ATP substrate

plasma membrane

NBF

TMD TMD

ADP + P

NBF NBF

TMD TMD

???

Structure of the ABC exporter Sav1866 from Staphylococcus aureus with bound nucleotide (Dawson and Locher 2006). Source: http://en.wikipedia.org/wiki/ATP-binding_cassette_transporter. Most exporters in prokaryotes, such as the multidrug exporter Sav1866, are made up of a homodimer consisting of two half-size transporters.

What is Lr34 doing?

• Lr34 confers resistance against multiple obligate biotrophic pathogens.

• Lr34 is associated with reduced intercellular hyphal growth but not with a hypersensitive response or papilla formation.

• The level of Lr34-mediated resistance for leaf rust infection correlated with the development of leaf tip necrosis.

These observations suggest that Lr34 could be due to a general physiological effect.

Microarray studies revealed that a similar set of genes was up-regulated in uninfected flag leaves of Lr34 containing near-isogenic lines and senescing flag leaves.

A. Senescence-related resistance?

Leaf tip necrosis in Lr34 containing lines

HvS40: a barley gene that is up-regulated during leaf senescence

Nonfluorescent chlorophyll catabolites (NCC): hallmarks of leaf senescence

Lr34 regulates senescence-like processes in the flag leaf of resistant plants.

Transcriptomics on Lr34 lines

Two transcriptomic studies on uninfected and infected wheat leaves with or without Lr34 were made (Hulbert et al. 2007, Phytopathology; Bolton et al. 2008, MPMI) Main conclusions: 1.  After infection, there is a high demand of cellular energy (increased carbon flux) 2.  Expression of defense genes was often higher in resistant plants: is Lr34 involved in priming of defense responses?

B. Resistance priming BEFORE infection?

PEN3/PDR 8 contributes to non-host resistance to inappopriate pathogens (Stein et al, Plant Cell 2006)

Pen3 mutants of Arabidopsis were more susceptible to infection with barley powdery mildew, pea powdery mildew and potato late blight.

Lipka et al., Current Opinon in Plant Biology, 2008

C. Transport of an antimicrobial metabolite?

From the FIELD To the FIELD!

Acknowledgments

University of Zürich CSIRO Canberra, Australia Liselotte Selter Joanna Risk Simon Krattinger Evans Lagudah Thomas Wicker Chauhan Harsh

IPK Gatersleben Jochen Kumlehn Götz Hensel

This work was supported by the European Research Council, GRCD and the Swiss National Science Foundation