projects

The soybean –P. sojae interaction

The major goal of the lab is to understand the molecular basis of the soybean-P. sojae interaction. As a first step towards understanding the interaction the lab has applied a map-based cloning strategy to isolate the disease resistance gene Rps1-k and Rps6. From high density and high resolution mapping of the Rps1 region, it was observed that Rps1-k mapped to one end of the introgressed region that was transferred from the cultivar Kingawa to Williams 82 through several generations of backcrossing. Screening of BAC libraries for markers linked to Rps1-k indicated that BAC libraries are highly under-represented for the Rps1-k region. Molecular markers mapped at 0.5-0.7 cM distances from Rps1 are well represented in BAC libraries. This indicates that the Rps1 locus is probably composed of repetitive sequences that are unstable in Escherichia coli.

Sequences of the end of BAC160, close to Rps1-k, showed homology to leucine-rich repeat regions (LRR) of disease resistance genes. The lab has applied this LRR sequence to identify BAC43, which mapped to the gap of the contigs in the Rps1 region. This BAC carries several LRR-like sequences. One copy has been sub-cloned and sequenced. Sequence data indicate that this particular copy has high identity to NBS-LRR type disease resistance genes such as I2C-1 gene, a homologue of which confers resistance of tomato to Fusarium oxysporum f. sp. lycopersici. BAC clones BAC18 and BAC99 that overlap with BAC43 were identified, and it was shown that BAC18 overlaps with BAC160. Sequencing of BAC18, 43, 99 and 160 indicated that these clones carry only one class of NBS-LRR type resistance genes. Susceptible soybean cultivar Williams 79 (rps1-k) has been being complemented with candidate NBS-LRR-like sequences fom the Rps1 region to identify Rps1-k. We have generated transgenic soybeans for two candidate genes.

The lab has initiated a map-based cloning project to isolate the complex locus that presumably carries Rps4 and Rps6. Both of the genes are temperature sensitive and fail to function at elevated temperatures. Recently the lab has shown that the Rps4 is highly unstable.

Signal transduction pathways in
the expression of non-host resistance

Non-host resistance, an inherent mechanism that presumably makes plants resistant to most microorganisms, has great potential in protecting crop plants against their pathogens. The lab intends to carry out a mutant screen for susceptible Arabidopsis genotypes against P. sojae. Both EMS and fast neutron mutants will be screened to achieve this goal.

Phosphoinositide signal pathway in plants

Phosphoinositides have been shown to be important signal molecules. This pathway has been shown to be essential for normal vision in fruit flies, for example. Most components of the mammalian phosphoinositide-signaling pathway have been found to have structural/functional equivalents in plants. The lab has applied molecular genetic approaches towards understanding the possible role and regulation of this signal pathway in plants. Previously, the had cloned a gene that encodes a plasma membrane-associated phosphoinositide-specific phospholipase C (PI-PLC) enzyme, a key regulatory enzyme of this signal pathway. Activated PI-PLC cleaves its substrate phosphoinositide bisphosphate into two signal molecules, inositol trisphosphate (IP3) and diacyl glycerol. IP3 has been shown to be involved in releasing Ca2+ that acts as a signal molecule in controlling many vital cell functions, including DNA replication.

Possible PI-PLC functions

The lab has demonstrated that IP3 content is increased in soybean cell suspensions by replenishing the growth medium with fresh nutrients. This nutrient-induced IP3 content is correlated with DNA replication, which indicates a possible role of this signal pathway in cell division or growth. Following infection, the resting state IP3 concentration is reduced in soybean cells. This may imply that the cell metabolites, presumably utilized for example in DNA replication, are diverted to meet the needs of host-pathogen interactions by reducing the PI-PLC activity.

To determine the possible functions for this signal pathway the lab has isolated T-DNA or transposon tagged plc mutants in Arabidopsis. The goal is to identify mutants for each of the PI-PLC genes and to incorporate all these mutations in a single plant by crossing. Such a plant most likely will fail to function normally, and will be very useful in identifying possible functions for this signal pathway. We have isolated two mutants, atplc1f and atplc2. AtPLC2 is constitutively expressed in Arabidopsis. The putative atplc2 mutant has shown retarded growth and early flowering. Whereas, atplc1f mutant has shown late flowering. AtPLC1F is expressed at the flowering time, and AtPLC2 probably partially complements the AtPLC1F-specific function in the atplc1f mutant. Currently the lab is carrying out complementation analyses for these mutants.

1. Positional cloning of the Phytophthora resistance gene Rps1-k

Phytophthora root and stem rot disease, caused by Phytophthora sojae, is the second most prevalent disease in soybeans. We are interested in cloning the most important and stable Phytophthora resistance gene, Rps1-k, by applying a map-based cloning approach. Cloning this gene should allow us to engineer soybean lines with stable and broad spectrum resistance against most, if not all, rapidly evolving P. sojae races or isolates.

Ms. Hongyu Gao is currently involved in sequencing ~200+ kb contig that carries several candidate gene sequences for Rps1-k.

Dr. Narayanan N. Narayanan has recently joined the lab to carry out the complementation analysis of these candidate genes.

Dr. Dipak Santra is involved in characterizing the genetic materials to identify possible functional Rps1-k-like genes in the Rps1 region. We speculate that many functional Rps genes have been conferring stability to Rps1-k for the last two decades.

This project has recently been funded by USDA-NRI. We presented a poster based on this project at the Plant, Animal, and Microbe genome meeting, held in San Diego, CA from January 12-16.

2. Construction of a YAC library

A yeast artificial chromosome (YAC) library to facilitate cloning of Rps1-k and Rps6 is being generated. This library will greatly complement the effort to construct a physical map of the soybean genome using bacterial artificial chromosome (BAC) libraries. A good physical map is the first step toward sequencing the whole soybean genome, as well as for cloning genes based on their map positions.

Dr. Dipak Santra initiated this project in October 2000.

Dr. Santra has brought the YAC cloning technology to ISU from Dr. Tom Tai’s lab in Stuttgart, Arkansas.

He has recently generated ~7,000 YAC clones carrying soybean DNA molecules.

ISPB has funded this project for three years (2000-2003).We presented a poster based on this project at the annual Plant, Animal, and Microbe genome meeting, held in San Diego, CA from January 12-16.

3. Characterization of the complex locus that carries
Rps4 and Rps6

We have recently isolated disease resistance gene-like sequences that co-segregate with the Rps4 gene. Detailed analysis of this locus indicates that the sequences undergo rapid rearrangements at very high frequencies (0.5-5%).

Ms. Hongyu Gao initiated this project.

We have collaborated with Dr. Silvia Cianzio to develop segregating materials and several dozens of F1s.

We have shown through DNA blot analyses that the sequences undergo rearrangement just before micro-sporogenesis.

We speculate that this rearrangement phenomenon may be involved in generating new variation in soybeans and other self-pollinated crop species.

Dr. Devinder Sandhu has also contributed significantly to this project.

A poster based on this project was presented at the annual Plant, Animal, and Microbe genome meeting, held in San Diego, CA from January 12-16.

4. Cloning of coding sequences of the soybean genome

The soybean genome is highly complex. It is ~10 fold larger than the Arabidopsis genome. Therefore, sequencing of the soybean genome is highly impractical, and it is unlikely that we will have the genome sequenced soon. The availability of protein coding sequences is necessary for many molecular biological analyses such as gene cloning through proteomics or determining map positions of genes. We are now evaluating an approach to clone coding sequences of the soybean genome .

Dr. Dipak Santra has developed a vector and cloned known DNA sequences in this vector (~2-6 kb) in Escherchia coli to evaluate the feasibility of this approach.

The success of this project should greatly expedite the soybean genomics as well as molecular biological research.

5. Engineering of soybean lines with stable and broadspectrum resistance against Phytophthora and other soybean pathogens

Our long-term goal is to develop soybean lines with genetically engineered stable and broadspectrum resistance against most pathogens. It has been shown in rice and Arabidopsis that overexpression of the NRP1 gene confers broadspectrum resistance against multiple pathogens.

Dr. Made Tasma has recently cloned the two soybean homologues of NPR1.

Dr. Tasma is currently characterizing these genes that are identical to Arabidopsis NPR1.

He will be complementing Arabidopsis npr1 mutant plants in the near future to determine the function of the soybean NPR1 homologues. The soybean gene that complements the NPR1-specific function in the npr1 mutant will be used to overexpress in transgenic soybean plants.

Transgenic plants will be evaluated for broadspectrum resistance against selected soybean pathogens, including P. sojae.

6. Identification of Arabidopsis non-host resistance genes that confer reistance against soybean pathogen P. sojae

Non-host resistance genes presumably confer durable and broadspectrum resistance against many non-pathgens. Therefore, such non-host resistance genes could be extremely valuable in designing crop species resistant to many pathogens. We have initiated a project to clone Arabidopsis genes involved in conferring resistance against soybean pathogen P. sojae. If we are successful in cloning such elusive non-host resistance genes, we will engineer soybean lines containing these genes to improve their resistance against an array of pathogens.

Dr. Made Tasma has recently shown that Arabidopsis seedlings can be pretreated with the soybean compound daidzein to attract P. sojae zoospores in numbers sufficient for successful infection.

He is currently evaluating the possibility of using a reporter system to isolate Arabidopsis mutants that fail to confer resistance against P. sojae race 1. In this reporter system, a firefly gene encoding luciferase is fused to a promoter that is activated following infection.

Plants showing higher levels of luminescence will be considered as putative mutants.

By carrying out progeny testing, mutants will be identified and used to clone non-host genes by applying a positional cloning approach.

7. Characterization of the phosphoinositide signal pathway in plants

We have previously cloned genes encoding the phosphatidylinositol-specific phospholipase Cs (PI-PLC) from soybean. This enzyme regulates the phosphoinositide signal pathway, which may be involved in regulating the second messenger Ca2+. We have reported biochemical evidence, which suggest that this enzyme up-regulates the DNA replication in soybean cell suspension following treatment with MS salts.

We have identified all nine PI-PLC genes from Arabidopsis. We have proposed to apply a reverse genetic approach to determine the function for all nine AtPLC genes.

We have identified two atplc mutants, atplc2 and atplc1f.

Ms. Junli Ji is in the process of complementing these mutants.

Current experiments include determining the transcript profiles for these nine genes by applying the RT-PCR approach.

Dr. Made Tasma is carrying out the RT-PCR experiments.

We hope that the results obtained from this research will complement our efforts to genetically modify soybeans for improved yield.