Maine Potato Board
Project Report (2006)
R-genes and aphid resistance in Solanum species
Benildo G. de los Reyes (PI) and
Andrei Alyokhin (Co-PI)
Department of Biological Sciences,
University of Maine-Orono
Executive Summary
Insect herbivores like the green peach aphid (GPA) and
potato aphid (PA) feed on their host plants by sucking photoassymilates from
the phloem sieve elements. With this mode of feeding, aphids not only inflict
physiological damage and considerable fitness cost to the host plant but they
also transmit disease-causing viruses including potato leaf roll virus and
potato virus Y. Leaf roll virus is vectored only by potato-colonizing aphid
species. R-genes are the major mediators of ‘gene-for-gene’
type disease resistance in higher plants. R-genes occur in multiple copies
often in tandemly duplicated arrays in the plant genome. Related studies in
tomato have shown that in addition to the disease-resistance function of
R-genes, they also play major roles in mechanisms of resistance to aphids and
nematode. Solanum bulbocastanum and other tuber-bearing wild species are
important sources of novel R-genes that confer broad-spectrum diseases
resistance. However, the roles of the specific R-gene analogs in insect defense
mechanism have not yet been explored. This research explores the wild Solanum germplasm as potential sources
of novel aphid resistance genes (including R-genes) using a combination of
phenotypic, physiological, molecular genetic and genomic approaches. The redefined (more focused) long-term goals
of this project originally established in the 2006 proposal are as follows:
1) To
conduct comparative evaluation of green peach aphid (GPA) and potato aphid (PA)
resistance among wild accessions of Solanum,
selected potato cultivars and transgenic-Katahdin plants expressing known R-genes
from S. bulbocastanum.
2) To
perform comparative analysis of the aphid-induced transcriptomes of resistant Solanum species and representative
susceptible potato genotype(s).
3) To
identify other candidate genes/loci involved in aphid resistance of wild Solanum species by molecular genetic
mapping.
The fist objective (phenotypic
evaluation and screening) was addressed in 2006. All commercial cultivars
included in this study were very susceptible to both GPA and PA. In contrast,
different wild Solanum species
exhibit different degrees of resistance to both GPA and PA, indicating that resistance
mechanisms have been conserved in the wild gene pool but were lost in the
cultivated species during the process of domestication. Two accessions of S. bulbocastanum (Acc. 283096, 243510)
in particular were found to be highly resistant to GPA and PA based on both
aphid fitness and host plant reaction experiments. These plants will be used as
models for genetic and molecular dissection of resistance mechanisms.
Project Accomplishments
The project was initiated in summer 2006 with objective #1
as the main focus. The first phase involved the development of a reliable
protocol for potato aphid (PA) and green peach aphid (GPA) resistance screening
and evaluation. Our initial aim was to develop a robust screening and
evaluation protocol that is fast, cheap and suitable for molecular genetic
experiments that require screening of large number of individuals in segregating
populations. Two different methods were evaluated for their suitability to the
requirements of the project. The first method involves the use of mini-cages (clip-on
cage method) made of plastic transparent tubing and sealed with a nylon mesh.
These mini-cages were assembled on aphid-inoculated leaf of a potted potato
plant. The second method involves growing potato shoots to 3 to 5 leaf stage in
hydroponic medium (0.25x Hoagland’s). The hydroponic potato shoots were
inoculated with a single adult aphid and enclosed in transparent plastic cages,
where aphid reproduction and survival can be monitored relatively easily on a
regular basis without disturbing the plant. In both methods, rates of aphid
reproduction and adult survival were monitored over a period of 1.5 months or
until the host plant dies. The clip-on
cage method caused some physiological distress to the plants (e.g. senescence
and wounding), thus was not pursued further. On the other hand, the hydroponically
grown potato shoots exhibited high vigor and were able to maintain the aphid
culture during the course of the experiment without the physical disturbance
observed in the clip-on cage method hence was used to continue the germplasm survey.
The results of our parallel screening and survey conducted in Island Falls (Sue
Ballou) and Orono (Ananya Mukherjee) are summarized as follows:
Solanum genotype
Figure 1.
Evaluation of cultivated potatoes and selected wild species accessions for
resistance to potato aphids (Island Falls experiment). Caged hydroponic potato
and wild potato shoots were inoculated with a single adult potato aphid at the
beginning of the experiment. Aphid reproduction on the specific host-plant
genotype was monitored by counting the number of new nymphs produced every
other day. Adult aphid survival on the specific host-plant genotype was also
monitored and recorded every other day. Monitoring was continued until the host
plant dies or until all the aphids are dead (in the case of tolerant genotypes).
Solanum tuberosum (1 = Katahdin; 2 =
Red Pontiac; 3 = Russet Burbank; 4 = AF1358; 5 = SP1144; 6 = SP1138; 7 =
SP904); S. commersonii (8 = Acc.
274833; 9 = Acc. 472834); S.
bulbocastanum (10 = Acc. 243505; 11 =
Acc. 243510; 12 = Acc. 283096); S.
oplocense (13 = Acc. 545870; 17 = Acc. 473188); S. boliviense (14 = Acc. 265860); S. demissum (15 = Acc. 218047; 16 = Acc. 473520); S. tarijense (18
= Acc. 458394); S. chacoense (19 =
Acc. 320287); S. polytrichon (20 =
Acc. 184773); S. trifidum (21 = Acc.
255541).
Relative fitness of PA on different
host Solanum genotypes . Solanum
species exhibit extensive genetic variation for resistance to PA with S. bulbocastanum showing the highest
level of resistance among the potato genotypes and wild species included in the
survey. Results of the PA screening is summarized in Fig. 1 (Island Falls
screening). Based on relative differences in aphid reproduction and adult
survival rates on the individual host plants, Solanum genotypes can be classified into distinct groups according
to their relative susceptibility to PA (groups arranged in increasing
susceptibility): Group 1, least
susceptible (bulbocastanum 283096)
< Group 2 (bulbocastanum 243510) < Group
3 (commersonii 274833, 472834; chacoense 320287) < Group 4 (polytrichon 184773) < Group
5 (oplocense, 545870, 473188; demissum 218047, 473520; tarijense 458394; trifidum 255541) < Group
6 (Red Pontiac, Russet Burbank, AF1358) < Group 7 (bulbocastanum 243505) < Group 8, most susceptible
(Katahdin).
Among the wild species tested, S. bulbocastanum is not only the most tolerant but it also exhibits
the widest variation among accessions. For instance, Acc. 283096 and 243510
(Groups 1 and 2) were found to be very tolerant while Acc. 243505 (Group 7)
exhibited a susceptibility level comparable to the most susceptible Katahdin.
Another interesting aspect of this study is the finding that three transgenic
lines with Katahdin genetic background (SP1138, SP904) appeared to show partial
resistance as suggested by the low aphid reproduction rate on these host
plants. SP1138 and SP904 are essentially genetically identical to Katahdin
except for a single transgene locus encoding two members of an R-gene cluster
introduced from chromosome-8 of S.
bulbocastanum (243510). These results are consistent with our initial
hypothesis that novel R-genes from exotic species might be playing important
roles in aphid resistance mechanisms. We are currently confirming these results
with further genetic and biochemical tests.
In contrast to SP1138 and SP904, which showed partial resistance the
other Katahdin transgenic line (SP1144) carrying the RB gene for late blight
resistance from S. bulbocastanum
showed similar level of susceptibility with the non-transgenic Katahdin,
suggesting that the RB gene does not play a role in resistance to insect
herbivores.
Figure 2. Evaluation
of differential host plant reaction to potato aphid infestation (Island Falls
experiment). Potted plants of each potato genotype and wild species in cages
were inoculated with 10 to 15 adult aphids at the beginning of the experiment.
Symptoms of physiological distress were monitored weekly. The susceptible
Katahdin died after less than 40 days after inoculation while S. bulbocastanum remained either
partially or completely healthy within the same duration of exposure to
PA. S.
trifidum, which is less susceptible to PA than Katahdin survived the 40
days exposure to PA but exhibited the classic symptom of physiological distress
associated with aphid injury (severe senescence).
Host plant reaction to PA
infestation. In
order to confirm the results of the PA fitness
experiments, we also monitored the differential survival rates of the
individual host plant genotypes under heavy aphid infestation (caged) for a
period of 45 days after inoculation (screening performed in Island Falls). An
example of the results from this experiment is shown in Fig. 2. The two
accessions of S. bulbocastanum, which
were found to be highly (283096)
and moderately (243510) resistant to PA showed a resistance reaction consistent
with the results of the aphid survival and reproduction experiments (Fig. 1).
The susceptible Katahdin died within 30 days after inoculation while S. bulbocastanum survived with either
limited or no visible signs of physiological distress (e.g., senescence,
wilting) associated with aphid injury.
GPA resistance of S. bulbocastanum. We also studied the reproductive rate and adult survival rate of green peach aphids (GPA) on different host Solanum species using the caged hydroponics method. In order to determine the relationship between PA and GPA resistance, subsequent experiments were focused on comparative analysis of GPA fitness on S. bulbocastanum (PA resistant) and S. tuberosum cv. Katahdin (PA susceptible) hosts. Results of this study (Fig. 3) showed S. bulbocastanum as the most resistant species to GPA among the potato genotypes and wild species included in the survey. Resistance is indicated by the relatively low GPA reproduction and adult survival, which mimics the pattern observed in the PA experiment. S. bulbocastanum Acc. 243510 showed the highest level of resistance. As expected both the non-transgenic and transgenic Katahdin line with the late blight resistance R-gene (RB) from S. bulbocastanum were very susceptible to GPA.
Solanum
genotype
Figure 3. Evaluation of cultivated potatoes and selected wild species accessions for resistance to green peach aphids (Orono experiment). Caged hydroponic potato and wild potato shoots were inoculated with a single adult GPA at the beginning of the experiment. Aphid reproduction on the specific host-plant genotype was monitored by counting the number of new nymphs produced every other day. Adult aphid survival on the specific host-plant genotype was also monitored and recorded every other day. Monitoring was continued until the host plant dies or until all the aphids are dead (in the case of tolerant genotypes). S. tuberosum (1 = Katahdin; 1a = Katahdin/SP904); S. demissum (2 = Acc. 473520); S. bulbocastanum (3 = Acc. 283096; 4 = Acc. 243505; 4a = Acc. 243510); S. commersonii (5 = Acc. 274833); S. tarijense (6 = Acc. 458394).
Summary of First Year Results and Future
Direction
The experiments we have conducted so far established two
accessions of S.
bulbocastanum as important sources of genes that can be used for the improvement of the resistance of the cultivated potatoes to aphids. Resistance was established based on relative aphid fitness on specific hosts and host-plant reaction evaluation. S. bulbocastanum is sexually incompatible with S. tuberosum. However, introgression of genes from this species to the cultivated potato is possible by classical biotechnological approach such as somatic hybridization or with more modern molecular strategies such as genetic engineering. In order to move forward to either of these two possible directions, questions related to the various genetic components and biochemical processes that determine resistance mechanisms need to be answered, and this will be the central focus of the project from year 2 and onwards. These questions will be addressed using forward genetic and functional genomic tools that are currently available in the potato research community.
Update on Previously Funded Project
The Maine Potato Board (MPB) has previously funded the PI on
a project that aims to understand the molecular basis of natural genetic
variation for abiotic stress tolerance in the genus Solanum. From the results of this project, which was funded in 2005
it was established that S. commersonii
is an excellent model for the investigation of stress tolerance mechanisms. The
information generated from this research established the current emphasis of this
project on understanding molecular mechanisms by functional genomics. In April
2006, this project was awarded a ‘material grant’ by The Institute of Genomic
Research (TIGR). The grant will allow the PI to develop comparative transcript
profiles of S. commersonii
(freeze-tolerant) and S. tuberosum
(cv. Red Pontiac) during a 2-week period of acclimation to low temperature. The
funded project will facilitate the identification of gene networks that could
lead to further identification of the critical regulators of stress response
signaling pathway. Potential
application of candidate genes to stress tolerance breeding will also be
explored.