Update:
The soybean cyst nematode (SCN) is a devastating pathogen that is responsible for over a billion dollars’ worth of yield losses in USA. 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. Devising novel management plans for this pest is essential, for which developing a deep understanding of host-pathogen molecular interactions is critical. Such knowledge will identify vulnerable points in host-pathogen interactions that can be manipulated in order to develop novel sources of nematode resistance in plants. Keeping this scenario in mind, we followed a two pronged strategy. Our first strategy focused on identifying novel effectors, the protein molecules produced and secreted by the cyst nematodes that are at the forefront of the host-pathogen interactions, while in the second strategy, we characterized individual effectors to identify their unique roles in parasitism. The long term of goal of our study is to unravel a complete effector repertoire of SCN which eventually will help us understand virulence differences shown by different SCN populations and to develop an in-depth understanding of nematode parasitism that can be exploited to identify novel resistance resources.
To achieve our first goal, we have been optimizing a novel system of mining SCN effectors from isolated esophageal gland cells, based on a previously reported method published from our lab (Maier, et al., MPMI, 2013). This ground-breaking method is designed to minimize RNA contamination from the surrounding tissues to identify even low level expressed effectors. As we have described below, one of our effector characterization studies has revealed that the nematode needs to carefully control effector abundance in root tissue suggesting even lowly expressed effectors could be playing crucial roles in parasitic success and identifying and characterizing such effectors is critical. Although our earlier published method of isolating RNA from the extracted secretory glands was already yielding useful quantity of RNA, we realized that the quality and quantity of this RNA pool was not ideal to reveal low level expressed genes. Thus, initially we focused our attention on optimizing our protocol. Our efforts revealed that the natural degradation of total RNA from the ethanol-fixed gland cells was one of the prime reasons for low RNA quality in our protocol. In order to solve this problem, we optimized our RNA collection method by altering chemical composition of buffers and tried multiple commercially available kits. Specifically, we were able to improve upon the quality of the RNA collected, largely through the isolation of individual gland cells directly into RNA extraction buffer. We found that by backfilling the microaspiration needle with extraction buffer from the PicoPure RNA Extraction kit, the gland cells were immediately lysed and the RNA was stabilized. Painstakingly following our optimization procedure, we have finally developed a reliable and robust protocol following which we can consistently produce high-quality RNA-sequencing libraries that yield a complete set of known SCN effectors of a given developmental stage. In addition, these libraries are yielding a large number of previously undiscovered effector candidates that we are working toward validating. Given the fact that these libraries are yielding both a complete suite of previously verified effectors and a vast number of novel candidates, we feel confident about usefulness of this system. It should be noted that this system has been aided greatly by the recent availability of the completed SCN genome, also an in-house development funded by NCSRP. Future plans for this technology will be to extend it to other SCN life-stages to create the complete suite of effectors, across all life stages, and populations.
Simultaneously, we have also been focusing our efforts on effector characterizations. Previous research findings have established that cyst nematodes re-differentiate plant cells at the infection sites and develop completely unique feeding structures called syncytia. The cyst nematodes need to maintain viable syncytia for a significant duration of time as they are their sole food source. Thus, successful establishment of a syncytium and sustaining its viability for a long duration is absolutely essential for nematode survival and reproduction. On the other hand, nematode resistant soybean cultivars have been shown to upregulate their defense responses and actively induce cell death at the site of infection to disrupt the nematodes’ attempts to establish viable syncytia to starve nematodes. Unfortunately, virulent SCN populations that can overcome host defense responses are spreading. Identifying and characterizing effectors that play roles in host defense suppression will help us better understand these complex molecular interactions. One of our effector characterization studies, regarding effector 4E02, has precisely achieved this and helped us further our understanding about the nematode-driven host defense suppression. For our effector characterization studies we utilized the model plant system Arabidopsis. Our previous research has shown that Arabidopsis-Heterodera schachtii (the sugar beet cyst nematode) is a very reliable and robust pathosystem that offers solid advantages for conducting experiments necessary for in-depth molecular analysis. The 4E02 effector is specifically expressed within the subventral esophageal gland cells. Since we observed its expression throughout all the life stages, we hypothesized that 4E02 has a role in defense modulation. To characterize potential 4E02 virulence functions, we generated stable transgenic Arabidopsis lines constitutively expressing the effector gene. To assess the effector’s roles in defense suppression, we assessed expression levels of six known defense-related plant genes in the transgenic lines. We observed statistically significant changes in expression levels of three defense-related genes. These data indicated that 4E02 indeed is involved in altering host defense pathways. Next, we analyzed whether the differential expression of the known defense genes is accompanied by changes in susceptibility to the sugar beet cyst nematode H. schachtii. Using our established nematode infection protocol, we observed that these transgenic lines are 22% more susceptible to the sugar beet cyst nematode compared to control plants, which supported the hypothesis that the 4E02 effector is a plant defense modulator.
Next we conducted experiments to identify host proteins targeted by this effector. Identifying protein interactors from plants can suggest cellular signaling networks impacted by effectors and can help describe its “mode of action”. To identify host interacting proteins, we conducted yeast 2-hybrid analysis - a procedure that allows studying protein interactions.
Our analysis revealed that the 4E02 effector interacts with the protein called ‘Resistant-to-Dehydration 21A’ (RD21A; At1g47128), a papain-like cysteine protease (PLCP). This interaction is particularly interesting as it has been previously documented that RD21A has a strong pro-death function and plays a key role in programmed cell death (PCD), especially during plant-pathogen interactions. As stated earlier, being obligate sedentary parasites, cyst nematodes need to prevent PCD and maintain syncytium viability for long periods to complete their life cycles. Throughout this period, the threat exists that the host plant triggers PCD to terminate the feeding site to starve cyst nematode, which has been documented in nematode- resistant soybean cultivars. Therefore, local suppression of PCD in the developing syncytium of host plant appears to be vital for the successful establishment and maintenance of a feeding site. Hence, we hypothesize that 4E02 could be targeting RD21A to modulate its function and prevent or delay PCD. Hence, we decided to probe this protein-protein interaction in greater detail.
According to the earlier published reports, effector 4E02 and RD21A accumulate in distinct subcellular compartments casting doubts on the physiological relevance of the protein-protein interaction we documented experimentally, i.e., can these potential interacting partners interact in planta if they accumulate in different cellular compartments? We therefore used bimolecular florescence complementation (BiFC) as an in planta assay to scrutinize this interaction as this method also would reveal the cellular compartment where the interaction is taking place. For BiFC assays, two non-fluorescing YFP halves are individually fused to two interacting candidates. If these candidate proteins interact, the two YFP halves are brought into close physical proximity, thus reconstituting fluorescence.
These BiFC assays showed strong fluorescence in presence of 4E02 and RD21A proteins, thereby confirming physical interaction in planta. However, most interestingly, these experiments also revealed that this interaction occurs in the cytoplasm and the nucleus, two cellular compartments that have not been reported for RD21A. An explanation is that 4E02 causes altered localization of RD21A. Collectively, our data indicate that the 4E02 effector not only binds to RD21A in vivo but also causes this protease to accumulate in different cell compartments.
Because we observed and confirmed the binding of the 4E02 effector to RD21A, we examined if this interaction affected the enzymatic activity of the RD21A protease. To answer this question, we decided to use an activity-based protein labeling technique (ABPP). We extensively collaborated with an international research group that has experience of conducting such analysis. In nutshell, ABPP gathers information about the functional state of the enzymes rather than about their abundance. Obtained ABPP data indicated that the effector does not alter the enzymatic activity of RD21A. We, therefore, concluded that 4E02 causes relocalization of the RD21A protease in its active form into a distinct subcellular compartment, which is a significant discovery. It is highly likely that by changing the location of the RD21A protease, it is removed from its usual substrate and function. In addition, since we have shown that protease activity per se is not impacted, RD21A in its new location will target a different set of substrates and will have a different function. In other words, the parasitic strategy of the nematode appears to be to interfere with normal, presumably defense related, RD21A function with the effector 4E02 but also to use the RD21A protease now for a different, yet to be identified novel purpose.
To identify potential targets of RD21A, we performed another yeast 2-hybrid analysis. We were able to identify eight RD21A interacting proteins. Interestingly, all them have confirmed functions in either stress response, cell wall biology or carbohydrate metabolism.
We, therefore, compared the root cell wall composition of 4E02 transgenic plants with control non-transformed plants, and these analyses revealed significant differences in carbohydrate compositions. Thus, we concluded that re-localization of active RD21A caused by the effector 4E02 leads to significant modulation of plant immune responses and syncytium growth via alteration of cell wall properties. This is a significant discovery and we are in a process of publishing this in a high-impact, peer reviewed journal.
Along with the host defense suppression, massive changes in enormous numbers of host genes observed at the infection site is another poorly understood phenomenon. Although multiple reports have identified a suite of soybean genes that change their expression as a result of nematode infection, the mechanistic details about how nematodes achieve such massive changes in large numbers of genes are far from clear. It is a likely scenario that nematodes secrete a specific effector or a small number of effectors that directly target either host DNA or proteins associated with host DNA and alter expression of a large number of host genes in favor of parasitism. Our second effector characterization story sheds light on this same phenomenon. We have discovered that the 32E03 effector is localized to the plant nucleus, a prime compartment of gene expression. We also identified that it specifically interacts with two host proteins that are closely associated with and involved in chemical alteration of host DNA. We know that these chemical changes in the host DNA can have major effects on gene expression. We also discovered massive changes in the expression levels of a specific subset of genes called rDNA genes in the transgenic lines expressing the effector, supporting our hypothesize that the effector alters gene expression of a suite of host genes by specifically targeting DNA-associated proteins. Most interestingly, we discovered that the effector abundance in host tissue impacts the rDNA expression changes, indicating nematodes have to precisely control the effector abundance as too much or too little of the effector can have adverse effects on parasitism. This unprecedented discovery will have implications in long-term pest management as now we can begin devising strategies to interfere this effector’s mode of action and disrupt syncytium formation. More technical details of our discovery are given in the next paragraph:
The cyst nematode effector 32E03 when expressed in Arabidopsis plant is transported into the nucleus where it co-localizes and interacts and with two plant proteins, namely HDT1 (a plant-specific histone deacetylase) and FKBP53 (a histone chaperone). Both these proteins are closely associated with plant DNA and are involved in controlling expression of a specific set of genes known as ribosomal RNA (rRNA) genes. rRNA is the major component of the ribosome, the plant cell machinery for protein synthesis. Expression of effector 32E03 in Arabidopsis transgenic plants led to a significant reduction in activities of histone deacetylases. It has previously been shown that changes in the activities of these proteins eventually impact gene expressions. In subsequent experiments, we found increased levels of acetylation of histone H3 along the ribosomal DNA (rDNA) in Arabidopsis plants expressing 32E03 as compared to wild-type plants. Due to increased histone H3 acetylation along the rDNA, DNA was actively transcribed in both directions, which lead to synthesis of double-stranded RNA and biogenesis of small interfering RNAs that silenced the rRNA genes. As a consequence, expression of the rRNA precursor was significantly inhibited. These Arabidopsis transgenic plants expressing 32E03 exhibited reduced susceptibility to nematode infection and a stunted phenotype when compared to wild-type plants. On the other hand, when we analyzed the rRNA precursor levels at the feeding site of nematodes in infected plants, we found an elevated level of the rRNA precursors. We then compared the expression level of 32E03 in transgenic plants and identified a low 32E03 expression transgenic line. Interestingly, these transgenic plants did not reveal any morphological variation and were highly susceptible to nematode infection. We then chose to characterize the function of the 32E03 effector in these transgenic lines that was conducive to nematode parasitism. Although these low 32E03 expression transgenic plants also revealed reduced histone deacetylase activities and increased acetylation of histone H3 along the rDNA chromatin, these plants showed high expression of rRNA precursors as compared to wild-type plants. We plan to analyze the cause for increased accumulation of rRNA precursors in these low 32E03 expression plant. So far, the effector 32E03 function documented here reveals a novel and powerful mechanism for how a parasite alters host chromatin structure to achieve gene expression changes required for infection success.
The soybean cyst nematode (SCN) is a devastating pathogen that is responsible for enormous financial losses to growers. We are focusing on the molecular interactions between the soybean host and the pathogen to identify vulnerable points that can be manipulated in order to develop better nematode management strategies. Our research has two main objectives. In our first objective, we focus on identifying novel effectors, i.e., the protein molecules produced and secreted by the cyst nematodes that are at the forefront of the host-pathogen interactions, while in the second objective, we functionally characterize individual effectors to identify their unique roles during parasitism.
A few years ago, we devised a groundbreaking method to specifically stain and isolate the effector-producing secretory glands from plant-parasitic nematodes. We have been working diligently to fine-tune this method to extract high quality RNA from these stained and extracted gland cells. In the past 6 months, we have worked to optimize the library construction with new conditions of improved total RNA quality through extraction buffer protection and the use of updated commercial kits with more efficient rRNA removal. The new techniques have yielded RNA-seq libraries that contain the complete set of known SCN effectors for a given expression profile. Even more exciting news is that these libraries contain large numbers of new effector candidates that we are currently working to validate. By producing stage-specific RNA-seq gland cell libraries using the updated protocol, we will be able to generate the entire suite of known SCN effectors, as well as create a large pool of novel effector candidates with which we can mine for new effectors.
Simultaneously, we continued to characterize SCN effector 32E03, a crucial effector in cyst nematode parasitism. To characterize the function of 32E03 in plants, we stably expressed 32E03 in plants. Earlier, we reported that plants expressing 32E03 at high level showed a stunted phenotype and reduced susceptibility to nematode infection. When we dissected the mechanism of the defense response in these plants, we found decreased expression of a specific set of RNA molecules called as rRNA. This finding hinted that high rRNA level in host plant cells is crucial for nematode parasitism.
Subsequently, we identified a set of 32E03 expression plants that did not show any variation in their morphology and resembled the wild-type plants and showed high susceptibility to nematodes. In these 32E03 expression plants, we found a good correlation between the low level of 32E03 and abundant plant rRNA accumulation. It is noteworthy to mention here that our analysis of rRNA in wild-type plant roots enriched in nematode-induced syncytium also revealed a high abundance of rRNA. Taken together, our results indicate that a high accumulation of rRNA in plant cells is required to alter the physiology of plant cells to sustain nematode parasitism. We furthermore determined that the 32E03 effector functions mediating host chromatin modifications to achieve gene expression changes required for successful infection. Based on these discoveries, we are currently writing a manuscript for publication.