Patricia,

The citrus pathogen Huanglongbing (HLB) is carried by the small fly-like insect, the Asian citrus psyllid (ACP). The bacterial pathogen resides in the insect saliva of the ACP. Thus, when it feeds on healthy plant tissue, the bacteria can spread from plant to plant causing disease. HLB has been present in Asia for over 100 years and has now spread to many countries such as Africa and recently the U.S. There is no cure for this disease and wiping out entire orchards to stop its spread is costly to growers.

Small citrus growers in Vietnam have known for a long time that planting guava next to citrus can repel the psyllid (Rouseff, et al., and Zaka, et al.). As such, scientists have worked to determine the volatile differences between the two plants and to determine the repelling agent. We now know that sulfur volatiles have this repelling effect. Consequently, inducing the emission of sulfur volatiles in plants that do not typically produce sulfur volatiles, by way of plant engineering, is one way this could be applied in practice. Other methods (which could potentially cause much less regulatory concern) would be the planting of border plants in the field (which naturally or via engineering produce sulfur volatiles) or by using these plants as rootstocks for young or older (inarching) commercial citrus varieties.

In addition to HLB, there are many other citrus pathogens that potentially could be controlled in this same manner. Also, we have reason to believe that these same sulfur volatiles may have additional beneficial effects for the plant in combating disease. For example, they may be effective in combating fungal disease in tomato. Preliminary results show that sulfur volatiles can inhibit the fungus, Botrytis cinerea, grey mold which often ruins strawberries. Thus, sulfur volatiles may aid in improving shelf life of fruit post-harvest (unpublished).

In regards to harmful side effects, thus far, growers in Vietnam see no negative effects in non-target insect populations. Moreover, the sulfur volatiles emitted by the plant exist in trace amounts and quickly diffuse through the air. Instead, it is believed that sulfur volatiles are potent enough to repel insect in close proximity to the leaf, but not further out. Thus we would not anticipate negative health effects. Sulfur volatiles could accumulate in enclosed spaces (ex. greenhouse), but as of yet, no negative health effects of growing sulfur volatile producing plants, such as guava, in enclosed spaces have been reported. However, the harmful side effects of any added volatile agent would need to be fully tested before being implemented commercially.
—Elenor, Mitch, Hyrum
1. Rouseff, Russell L., et al. “Sulfur volatiles in guava (Psidium guajava L.) leaves: possible defense mechanism.” Journal of agricultural and food chemistry 56.19 (2008): 8905-8910.

2. Zaka, Syed Muhammad, et al. “Repellent effect of guava leaf volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus.” Insect Science 17.1 (2010): 39-45.

Consider the case of Xylella fastidiosa. This bacterium causes disease in a wide range of hosts, including citrus (Citrus variegated chlorosis), grape (Pierce’s disease), almond (almond leaf scorch), and a variety of different crops. [1, 2]. After becoming infected with X. fastidiosa the spread of this disease is controlled using pesticides and the destruction of the plant/surrounding plants. One of the compounds X. fastidiosa uses to communicate has been identified as a fatty acid known as 2(Z)-tetradecenoic acid ³. This compound could potentially be applied via a spray by growers of grape or citrus. However, one way we could integrate two of our approaches for greater power in order to control this disease would be the hybrid of “Boosting the Plant’s Immune System” and “Jamming bacterial communication.” Plants could be engineered to produce either more of this communication molecule, or an alternative compound that competes with the native compound to bind with the communication receptor. Either way, the plant itself would be acting to misregulate the bacteria. This would save time and money for the growers of these plants—savings which would then be passed on to consumers. Further, this would minimize harmful side effects of heavy pesticide use in the environment, as excess spray would not be accumulating in our water reservoirs. Some plants have been shown to naturally have products with these capabilities ⁴, and the idea of engineering the plant to innately defend itself in this way is currently being used by Steven Lindow’s lab at UC Berkeley to fight Pierce’s Disease (5). It is recognized that the key to durable (long-lasting) pathogen resistance in the field is to “stack” or combine different traits in order to control or modify various aspects of plant-pathogen interactions. Ultimately, multi-tiered approaches will probably be most effective in combating destructive plant pathogens. To take our three projects as an example, it might be possible to engineer a plant to produce volatiles that deter herbivores or insect vectors of pathogens (Elenor). In order to further improve plant defense, we might be able to introduce additional traits into this plant, such as the ability to produce anti-quorum sensing compounds (Hyrum) only when the plant recognizes pathogen infection (Mitch).

1. Hill, B.L.,et al., Populations of Xylella fastidiosa in plants required for transmission by an efficient vector. Phytopathology, 1997. 87(12): p. 1197-1201.
2. Chatterjee, S., et al., Living in two worlds: The plant and insect lifestyles of Xylella fastidiosa. Annual Review of Phytopathology, 2008. 46: p. 243-271.

3. Beaulieu, E.D., et al., Characterization of a Diffusible Signaling Factor from Xylella fastidiosa. Mbio, 2013. 4(1).
4. Adonizio, A.L., et al., Anti-quorum sensing activity of medicinal plants in southern Florida. Journal of Ethnopharmacology, 2006. 105(3): p. 427-435.
5. Lindow, E. L., et al., Enhancing control of Pierce’s Disease by Augmenting Pathogen Signal Molecules, Symposium Proceedings, Pierce’s Disease Research Symposium, 2011. P. 144-153.

Thank you, Hyrum.
Catherine

Hi Liliana, thanks for your interest. Most of my work has been done in tomato and the model plant Arabidopsis. However, due to the conserved nature of the plant immune system, it is highly probable that information gained in these systems will translate well into other important crops species. For example, a plant immune receptor (a protein called EFR which recognizes a bacterial molecule called EF-Tu) is only found in Arabidopsis and closely related species. EFR has been transferred to tomato and other plants through biotechnology and remarkably, it is fully functional in these distantly related species! It actually confers broad-spectrum disease resistance to multiple bacterial pathogens of these crops. Furthermore, because it is fully functional, it indicates that the downstream immune signaling components are conserved across a wide range of plant species. This finding also suggests that similar biotechnology approaches for enhancing disease resistance in crops is certainly feasible. Moreover, by engineering plants to be more resistant to pathogens, we can actually reduce the need for pesticide use and in turn, the harmful effects of heavy pesticide sprays on the environment. Here is a reference for further reading on this interesting topic:

Lacombe, Séverine, et al. “Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance.” Nature biotechnology 28.4 (2010): 365-369.

Some plants, like citrus, already produce specific types of sulfur volatiles. However, these compounds do not deter the insect vectors that we are trying to control. Because the basic biochemical pathways for sulfur volatile production are present in citrus, it is certainly feasible that we can “tweak” these pathways by introducing an enzyme that produces the volatile of interest in these plants. Specifically, citrus naturally produces the sulfur volatile dimethyl sulfide, but the mono sulfides show no repellent effects. Thus, we are working to change the biochemistry to produce the sulfur volatile dimethyl disulfide (di-sulfide) that show repelling effects to the insect that causes disease in citrus (Zaka, et al. 2010)

With regards to sulfur volatiles, we do not see any negative effects thus far. Growers in Vietnam have used guava plants that emit sulfur volatiles naturally as border plants to repel the insect carrying disease for many years. Thus far, we see no negative effects in regards to changing the food chain of insects. Moreover, the sulfur volatiles that plants emit are present at trace levels and quickly diffuse in the air. The idea is that sulfur volatiles are potent enough to repel insect in close proximity to the leaf, but not further out. In addition, not all insects are deterred by sulfur volatiles. For example guava and some broccoli varieties produce the volatiles we are interested in and this has not impacted their ability to be pollinated by beneficial insects.
-Elenor & Hyrum

Yes, in our opinion other bacterial communities could be effected by trying to misregulate the bacterial communication of pathogens. We are learning more and more that the microbiome of every living organism is effected in a number of ways by perturbations in the system. However, it has been found that many species of bacteria have very specific signaling compounds ¹. The ideal compounds will effect specific pathogen but not nonpathogenic species and be biodegradable.
1.Newman, K.L., et al., Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(6): p. 1737-1742.

Hi Alan, thanks for your question. You are right; my main project is “exploratory” in that I am using quantitative proteomics to identify and understand plant immune responses at the plasma membrane. I would like to emphasize that although my project would be considered more in the discovery phase of a plant biotechnology pipeline, it has already yielded some important outcomes. So far we have identified many proteins that are modulated during plant defense. I have used this information in conjunction with a reverse genetics approach to demonstrate that several of these proteins do indeed control plant immunity to pathogens (please see one example in Figure 4 of our poster). To extend this work, we are currently exploring the potential of using these genes to engineer crop plants like tomato to be more resistant to agricultural pathogens. As you alluded to, a key factor in plant biotechnology (or any biotech field) is to translate basic science discoveries into something that is beneficial to the public (e.g. farmers, consumers, etc.). I anticipate that my work will contribute to a framework for understanding an important aspect of biology (plant immunity to pathogens), but it also has direct applications to important agricultural problems (enhancing pathogen resistance).

Alan,
A large portion of the work with quorum sensing is also exploratory. We are developing a high-throughput screen to detect anti-quorum sensing compounds (compounds that jam the bacterial communication). However, interestingly, we can see that communication compounds are often very specific for bacterial species—meaning, even very closely related bacterial species many times do not share the same signaling compound (the changes are enough to cause only partial bioactivity). For example, a biomonitor strain of Xanthamonas campestris that brightly fluoresces upon the detection of its own communication compound shows greatly less fluorescence in the presence of Xylella fastidiosa’s communication compound (X. fastidiosa is so closely related that it was once considered Xanthamonas and the identified signaling molecules are very closely related biochemically) ¹. As such, when our screen identifies a quorum sensing (communication) compound or a compound that inhibits this communication (anti-quorum sensing) with “small” bioactivity, we expect that this compound may have large effects in an alternative bacterial strain.
Additionally, though most work has concentrated on carbon chains of different lengths, saturation, and side groups, it has recently been found that proteins can also serve as quorum sensing compounds ². We expect to find that many microbe associated molecule patterns (MAMPs), which the plant innate immunity system has used to detect bacteria, are in actuality detecting the bacterial communication signals.

1.Newman, K.L., et al., Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(6): p. 1737-1742.
2.Han, S.W., et al., Small Protein-Mediated Quorum Sensing in a Gram-Negative Bacterium. Plos One, 2011. 6(12).