Biotrophs on Arabidopsis
CRUTE at UK.AC.AFRC.EMRS
CRUTE at UK.AC.AFRC.EMRS
Mon Jun 22 07:33:00 EST 1992
Below is a summary of the progress reports that Ian and I (Eric)
contributed to the quarterly newsletter of the UK-AFRC Arabidopsis
initiative. Since the newsletter only had limited distribution, it
seemed worthwhile that we share our summary with more of the
Arabidopsis community via the network.
Although speaking with hindsight, writing the reports has most
definitely been a useful exercise to find out even for ourselves how
far we have come over the past two years. I hope this contribution to
the network will stimulate other groups in the community to provide
a similar report of their own work.
Identification and mapping of genes for resistance of Arabidopsis to
biotrophic fungal pathogens /Peronospora parasitica/ and /Albugo
Eric Holub and Ian Crute; HRI-East Malling, Kent.
Jim Beynon, Mahmut Tor and Edemar Brose, Wye College, Kent.
Investigation began of two obligate fungal biotrophs /Albugo
candida/ and /Peronospora parasitica/ which commonly parasitize
Arabidopsis in the UK.
Methods were developed for routine, experimental manipulation of
the two fungi on Arabidopsis.
Numerous Arabidopsis accessions were found which were resistant
to Peronospora as defined by the lack of asexual sporulation after
inoculation. Two fungal isolates (CALA1 and EMOY2) were used and
accessions were found which were resistant to one isolate or the
other; or to both isolates. Only one accession was found which was
resistant to Albugo.
Pollinations for a half-diallel cross of fourteen Arabidopsis
accessions was initiated to provide germplasm for investigating the
inheritance of resistance to Peronospora. The accessions used
includes examples of common laboratory lines as well as accessions
from locations where the isolates were collected.
A simple hypothetical model involving three resistance loci was
proposed to explain differential interactions between six accessions
from the half-diallel cross and four isolates of Peronospora.
Evidence was presented for expansion of the model. Two additional
loci for resistance were predicted to explain new differences
observed from microscopic examinations of the interactions,
tests made with new isolates, and a partial F2 analysis of the half-
diallel cross. Only six isolates had been tested to date and they
proved to be six different pathotypes. RFLP mapping of loci for
resistance to Peronospora was started by a postdoctoral researcher
Mahmut Tor in Jim Beynon's lab. And, mapping of a single gene for
resistance to Albugo was initiated by a PhD student Edemar Brose,
also in Jim's lab.
Further differentiation of phenotypic interactions between
Arabidopsis and Peronospora has been possible. Phenotypes
include: full susceptibility with sporophores profuse and visible 3
days after inoculation (dai); sporulation sparse to moderate and
delayed at least 48 h; no sporophores produced but flecking lesions
of necrotic host cells are visible to the naked eye 7 dai; and no
sporophores produced but necrotic "spots" or "pits" are visible to the
naked eye at 3 dai. Spots and pits are thought to be different
responses. Both are discrete epidermal lesions ca. 1 mm in diameter
3 dai and usually have a chlorotic halo, but pits involve more
pronounced cellular collapse forming a depression in the epidermis.
Both interactions are further characterized 7 dai as either
determinate or expanding.
The hypothetical genetic model now requires at least eight resistance loci
to explain interactions observed between six accessions and six isolates
of Peronospora. Nearly all F2 populations from crosses made between the
parents Weiningen, Nd0, Oy0, Col0 gl-1, La-er, RLD, Tsu0, and Keswick37
have now been tested against isolates CALA1 and EMOY2. Continued analysis
of the half-diallel cross will undoubtedly reveal new genes for investigation,
however, three of the putative loci already identified have been chosen
for investigation in more detail.
Four of the phenotypes described above segregate in F2 Col0 gl-1 x Nd0
following separate inoculations with isolates CALA1 and EMOY2. In response
to CALA1, the fully susceptible reaction of Nd0 segregates in the
cross together with the flecking phenotype of Col0. The flecking
reaction is probably controlled by an incompletely dominant allele at
a single locus. In response to EMOY2, the delayed sporulation
reaction of Col0 segregates in the cross together with the pitting
phenotype of Nd0. Pitting is controlled by an single completely
dominant allele at a different locus. The delayed sporulation is thought
to be controlled by a weak, yet genotype specific, allele. All four
phenotypes can be readily scored in the F3 generation so we anticipate
being able to determine the genotype at both loci (see Table), except
when "pitting" masks delayed sporulation.
The pitting allele (RPp1) and flecking allele (RPp2) are now our
prime targets for molecular analysis. With a stroke of fortune, we
observed linkage between the pitting allele and the glabrous-1 locus
so we have already located it to chromosome 3. Mahmut has since
confirmed the location of this allele and has located RPp2 to
chromosome 4 using a diagnostic set of RFLP probes provided by
Elliot Meyerowitz. It should also be possible to map the locus
controlling delayed sporulation of Col0 (RPp4) following inoculation
with EMOY2. RPp4 is closely linked to RPp2.
Resistance of Arabidopsis to Albugo is also being investigated in
a similar manner, however the system is less advanced. Nevertheless,
thirteen Arabidopsis accessions have been found on which
sporulation by Albugo does not occur or is delayed following
inoculation with an isolate collected from East Malling. Nine of
these putatively resistant accessions were collected from locations
in the UK and four from Germany. The responses include: no pustules
and no macroscopically visible necrosis; necrotic flecking without
pustules; discrete chlorotic patches without pustules; and delayed
production of small pustules. Crosses between plants selected from
these accessions have been made, and will be used to identify genes
for resistance to the East Malling isolate of Albugo. The first
resistance locus (RAc1) is being mapped in Wein (susceptible) x
Table. Representative sample of F2 Col x Nd0 genotypes predicted
from F3 progeny tests for segregation in response to Peronospora
isolates EMOY2 and CALA2.
F3 segregation 
F3 family EMOY2 CALA2 F2 Genotype
911448 0:0:0:10 0:0:0:12 r1r1 r2r2 r4r4
911455 0:2:8:0 0:12:1:0 r1r1 R2R2 R4R4
911449 0:3:5:2 0:3:6:3 r1r1 R2r2 R4r4
911430 14:0:0:0 0:0:0:10 R1R1 r2r2 ? ?
911525 12:0:0:4 0:0:0:10 R1r1 r2r2 r4r4
911469 10:2:0:0 0:0:0:10 R1r1 r2r2 R4R4
911470 7:5:1:2 0:0:0:10 R1r1 r2r2 R4r4
 Data (left to right) are numbers of plants in each of four
phenotypic classes: pitting necrosis of cotyledons 3 days after
inoculation (dai), no sporophores; necrotic flecks 7 dai, no
sporophores; sporophores absent 3 dai but sparse to moderate
7 dai; sporophores present and profuse 3 dai.
 Predictions for the status of two putative loci for
resistance to EMOY2. R1 is a completely dominant allele
from the Nd0 parent causing pitting necrosis to EMOY2; R2
is a mostly dominant allele from Col0 causing flecking to
CALA2; and R4 is an weakly dominant gene from the Col0
parent causing delayed asexual sporulation to EMOY2.
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