Update:
Objective 1) Select and incorporate genes expected to improve disease resistance, yield, and seed quality to provide breeding material for production of improved soybean cultivars.

We completed taking the third generation transgenic plants to seed to produce the fourth generation (T4) that were made to overexpress the WRKY and MYB transcription factor genes of interest. Approximately 100 – 150 seeds were collected from each of the plants. The T4 generation transgenic soybean plants were tested to identify lines that were homozygous for the transgenes of interest. The main assay used was to test for presence of the BAR protein, which is the herbicide resistance gene that is present in all the transgenic lines and is used to select for the presence of the transgenes. We selected at least two independent transgenic lines for each of the six transgenes of interest for analysis of disease resistance. Given the relatively low amount of seed, we decided to focus on one disease for which we have a relatively straightforward and robust assay. Approximately, 20 T4 seedlings of each transgene were inoculated in growth chambers with Bean pod mottle virus (BPMV) tagged with the green fluorescent protein (GFP). As a control, wild type Williams 82 seedlings were also inoculated, so that BPMV infection could be compared. Two independent replicates of the experiment were performed. Our expectation was that some of the lines would be less susceptible to BPMV, because some of the transgenes were associated with defense in previous studies. All seedlings were scored for symptoms, GFP fluorescence in systemic leaves, and we performed ELISA assays to quantify relative virus levels. We observed that none of the six transgenes conferred resistance to BPMV, suggesting that the plants did not have enhanced defenses against this pathogen. This result has discouraged us from testing for resistance to other pathogens. During this time, we have also developed a soybean drought-tolerance assay in the greenhouse. We hypothesize the four of the transgenes may have an effect on soybean responses to water stress. We did not have time to screen these lines for drought tolerance yet, but we plan to in the next year using other resources.

We continued to work with Dr. Ling Li in determining if the QQS gene from Arabidopsis enhances soybean defenses. We have demonstrated that soybean plants expressing QQS or overexpressing its interacting partner NF-YC4 are less susceptible to BPMV in growth chamber experiments in addition to Pseudomonas syringae pathovar glycinea (bacterial blight), which was tested previously. The results we obtained with Dr. Li suggest that the QQS gene could be useful in promoting soybean health as well as enhancing protein content of the seed.

Objective 2) Exploit soybean proteins targeted by SCN to develop SCN-resistance soybean.
The soybean cyst nematode (SCN) is one of the most damaging soybean pathogens that is responsible for enormous financial losses to growers. Combination of crop rotation, and use of SCN resistant soybean lines and nematicides is routinely used as a strategy to reduce yield losses due to SCN infestation but the costs and environmental hazards of current nematicides, limited financial benefits of crop rotation, and the emergence and spread of SCN populations that overcome the limited available sources of natural resistance are exacerbating the problem. Identifying vulnerable points in host-SCN interactions that can be manipulated in order to develop novel sources of nematode resistance in plants is essential for which conducting detailed analysis of the molecular interactions of host-SCN is critical. The major source of molecular signals sent by pathogens to their host is effector proteins. Conducting in-depth functional characterization studies of these effectors can shed light on the underlying molecular mechanisms of the strategies employed by pathogens to establish successful infection. Once such mechanisms are known, various biotechnology approaches can be followed to disrupt the same to develop pathogen resistance in host. Keeping this scenario in mind, we have been conducting in-depth functional characterization of various SCN effectors. One of the effectors that we have been studying in past few years is 1C01.
This effector has a well-characterized “acetyl transferase” domain. Using yeast 2-hybrid technique, we have determined that 1C01 interacts with a unique soybean cytoskeleton protein, named “clasp”, which is highly conserved across various species as well as a nucleus localized transcription factor. Literature suggests that “clasp” plays an important role during cell wall expansion. Since the nematode feeding site (syncytium) development involves rapid cell wall expansion, the interaction between the effector and “clasp” is of particular interest. We have confirmed this interaction in planta by using bimolecular fluorescence complementation (BiFC) approach in Nicotiana benthamiana. Most interestingly, we confirmed this interaction between the 1C01 effector from Heterodera glycines and soybean clasp as well as between the effector heterolog from Heterodera schachtii and Arabidopsis clasp, indicating this interaction is very specific and conserved across the species. This result also reaffirms feasibility of using Heterodera schachtii-Arabidopsis pathosystem to study soybean cyst nematode effectors. We continue to study the biological significance of this effector and its host interactors and their importance for nematode parasitism. We successfully cloned the effector homologs from both Heterodera glycines and Heterodera schachtii species as well as “clasp” homologs from both Arabidopsis and soybean into subcellular localization vectors. When these constructs were co-expressed transiently in Nicotiana benthamiana, we observed strong co-localization reaffirming the interaction between the effector and its host interacting protein for both pathosystems. We have also generated transgenic Arabidopsis lines constitutively expressing 1C01 from Heterodera schachtii using 35S promoter and we are testing them altered nematode susceptibility.

According to the published research reports, effectors with acetyl transferase domain from other plant pathogens are involved in defense suppression. We conducted defense suppression assay by expressing this effector transiently in Nicotiana benthamiana. Our results indicate that this effector robustly suppresses plant defense. It is already known that during the molecular host-nematode interaction, host upregulates defense responses at the sites of infection. The parasitic success of the cyst nematodes, in turn, relies on the robust suppression of these host defense responses. The functional characterization of the effector 1C01 is of particular interest as according our data it seems to be involved in defense suppression. With these results, we are determined to study this interaction even deeper and have established collaboration with another group that has worked extensively with clasp proteins. In the future, their involvement will be essential to pinpoint the functional role of clasp in SCN parasitism in general and in syncytium development in particular.

Objective 3) Accelerating the soybean reproductive cycle. Due to technical problems that were encountered in years 1 and 2, we were unable to test if we could accelerate the soybean reproductive cycle in different varieties or maturity groups.

Objective 1) Select and incorporate genes expected to improve disease resistance, yield, and seed quality to provide breeding material for production of improved soybean cultivars.

We made transgenic plants that overproduced six different transcription factor genes that are members of two families known as WRKY and MYB. These transcription factor genes had previously been identified in experiments as genes that might protect soybean against pathogens or drought stress. We hypothesized the overexpressing these genes would have a positive effect on soybean defenses. We developed multiple lines overexpressing each transcription factor, identified lines that were homozygous (carried two copies of the transgenes). The transgenic lines were then test for resistance to Bean pod mottle virus (BPMV), but none of them were more resistant to the virus. The reason for this is not clear, but one possibility is that we did not recover a soybean line that overexpressed the transcription factor enough to have an effect. We remain interested in further testing some of these transgenic lines for resistance to drought. We developed a drought stress assay for soybean in our lab and intend to pursue this in the future.

We also worked with a collaborating lab (Dr. Ling Li) on another transgene called QQS, which was originally found in the model plant Arabidopsis. The QQS was previously shown by Dr. Li to increase protein content of transgenic soybean seeds. Working with her, we also showed that the QQS transgene also helps to protect soybean plants from disease caused by viruses and bacteria. QQS is thus very interesting for its ability to manipulate soybean seed composition and enhance disease defense.

Objective 2) Exploit soybean proteins targeted by SCN to develop SCN-resistance soybean.
Heterodera glycines or the soybean cyst nematode (SCN) is one of the most devastating soybean pests and there is an urgent need to update current SCN management. Developing detailed understanding of the strategies employed by the SCN to parasitize its host is essential in order to develop novel resistance resources. At the molecular level, SCN communicates with its host via effectors, the proteins produced in and secreted by the esophageal glands. These SCN secreted effectors are thought to play key roles in the profound changes observed at the infection sites that culminate in feeding structures (the syncytia). In-depth functional characterization of these effectors can reveal their specific roles in SCN parasitism. Once we identify their roles and their host targets, one can devise strategies to disrupt these interactions and potentially develop novel resistance resources. Hence, during this funding cycle, we focused our attention on the functional characterization of the effector 1C01.

Being sedentary and biotrophic endoparasite, the SCN has to suppress host defense responses for a long duration in order to complete its life cycle. We discovered that 1C01, which also has a well-defined acetyl transferase domain, can robustly suppress plant defense responses thus making it an ideal candidate to study SCN driven host defense suppression. Most interestingly other researchers have established that the effectors from other pathogenic species with such domain also function as host defense suppressors. We have also identified its host interacting partner, a highly conserved cytoskeletal protein. We have confirmed this interaction using multiple molecular approaches. This grant has helped us tremendously to make this important research breakthrough and on the basis of these results we have established a thriving collaboration with another group that has worked on this cytoskeletal protein in detail. We are confident that this breakthrough will advance the knowledge of SCN driven host defense suppression considerably. Along with achieving our research goals, this grant has also helped train post-doctoral research associates in our group in molecular pathology assays, which has helped them build their careers.

Objective 3) Accelerating the soybean reproductive cycle. The plan was to use our Bean pod mottle virus (BPMV) vector to express a gene called FT in soybean plants. The FT gene causes plants to flower early, and therefore, the lifecycle can be greatly sped up. We made the BPMV designed to express FT and confirmed that it was correctly made. However, we were unable to show that the BPMV + FT could infect soybean plants and were unable to test if it could accelerate the soybean reproductive cycle. Based on successes in other plant species, we think the idea is feasible, but a different approach may be needed.