Application of RNAi-based methods to aphid vectors

Michaël Mulot, PhD student, UMR 1131 INRA–Univ. Strasbourg, Santé de la Vigne et Qualité du Vin, Equipe Virologie & Vection, F 68000 Colmar, France.

The genomes of several aphid species are now available, providing a tremendous opportunity to identify genes that have vital functions or are involved in the transmission of viruses. In this purpose, strategies based on RNA interference (RNAi) have been developed for aphids. RNAi is based on the detection of double-stranded RNA (dsRNA) and small interfering RNA (siRNA) molecules by insect cells. This detection can activate the degradation of the messenger RNAs of complementary sequences in the insect cells and thus inhibit the expression of the target gene. Various methods have already been employed to deliver such double-stranded RNA molecules in aphids, but no study comparing the efficacy of these methods was available until now.

In a recent study (Mulot et al., Viruses 2016, 8, 316, doi: 10.3390/v8110316), various methods based on the oral acquisition of double-stranded RNA molecules were used in the aphid vector Myzus persicae. Two genes expressed at different levels in the aphid were selected to conduct this study. This study is the first in which several RNAi methods have been evaluated in a single laboratory using the same genes, the same aphid clone, and the same evaluation criteria for RNAi efficacy, eliminating by this way a number of variation sources in the results. In addition, all methods targeted the same region of each gene under study. These comparative experiments showed (1) that the efficiency of RNAi may differ considerably depending on the target gene, and (2) that intestinal cells of the aphid appear to be preferentially affected by the mechanism of RNAi after oral acquisition of dsRNA. In addition, the use of plants infected with a recombinant Tobacco rattle virus (TRV, a plant virus which forms dsRNA-like replication intermediates) proved to be a promising method for inhibiting the expression of aphid genes, since it has many practical advantages compared to other approaches. This work stresses the need to continue developing innovative strategies to effectively inhibit the expression of aphid genes, e.g. those involved in virus transmission.

A nice review on plant virus vectors by Dietzgen et al. (2016)

Over the last few years, a number of very useful review papers on phytovirus transmission by insects and other vectors have been published, including the chapters in the recent book edited by J.K. Brown at APS Press (previously announced on this blog),  each review putting emphasis on various facets of the domain.

The review by Dietzgen et al. (2016) featured here is also very interesting, as it summarizes the most recent findings at the very cutting edge of research.  It contains a ref list of 177 items.  Moreover, it focusses on the parallels drawn between plant and animal virus vectors, leading the authors to discuss how the knowledge gained with animal virus vectors can stimulate the thoughts on the side of plant virus vectors, and opens new avenues towards better understanding the complex phytovirus–vector interactions at the cellular and molecular levels.  Finally, the review discusses the great potential of novel approaches, such as “omics” and symbiosis biology, in our favorite field of research.

Ralf G. Dietzgen, Krin S. Mann and Karyn N. Johnson
Plant Virus–Insect Vector Interactions: Current and Potential Future Research Directions.
Viruses 2016, 8 (11), 303; doi:10.3390/v8110303

PS  Other vector-related papers in the same journal: here.

Etienne Herrbach
INRA Colmar, France

Similarities to academia?

There is plenty of material available about the problems of evaluating science and scientists based on quantitative measures such as the impact factor of journals (and other measurements). In addition, there are more and more examples of individuals being interviewed for positions, or being promoted, using ‘achievements’ such as a TED talk or having a paper covered by WIRED magazine, as evidence that their work is good. There are many other examples of what I consider a distortion of how to evaluate science and the scientific literature. There is a lot of what I would call PR (public relations) in science today, just as there is in politics or whatever else. Maybe some of it is driven by the lack of funds, or twitter, but the fact is that things are done differently today.

Anyway, yesterday I read a piece in Vanity Fair that I thought was very interesting. The story is worth reading as it illustrates a bit how some of the biotech industry operates. But I cannot avoid thinking of parallels with my own professional life/experience.


RNAseq and B-side results

Phytoplasmas interact with their insect vectors in a very intimate way [1]. In order to deepen this relationship, with many unknown aspects, we performed an RNA seq project on the leafhopper Euscelidius variegatus, which is a well-known natural vector of aster yellows phytoplasmas, including chrysanthemum yellows phytoplasma (CY) and an efficient laboratory vector of Flavescence dorée phytoplasma (FD), as already mentioned in a previous post, see Phytoplasma and their vectors: more than just data flirting.
The RNAseq project originally aimed at investigating the interactions between the insect and the two phytoplasmas, exploiting the fact that the same species could transmit two phylogenetically distant phytoplasmas (competition of the two phytoplasmas in E. variegatus has also been investigated [2]). Main purposes were to provide clues on the insect response to phytoplasma infection and new insights on the molecular mechanisms used by phytoplasmas during insect colonization. However, valuable additional information about the composition of bacterial and virus population in E. variegatus was also obtained, perfectly in line with a recently discussed topic on this blog, see NGS-derived thought.
Among viruses we focused on a picorna-like virus, because we assembled and obtained a very long contig that probably represents the whole viral genome with a good read coverage. Infection was confirmed by RT-PCR in all the tested insects, so it can be considered endemic in the leafhopper colony. This finding was unexpected as the infection was apparently asymptomatic: insect fitness does not seem to be altered, at least in terms of longevity and prolificity. Although we have no experimental clues yet, the virus could be transmitted either transovarially or by a oro-fecal route, in accordance with the literature on other phylogenetically related arboviruses [3].
Interestingly, the virus was detected in both phytoplasma-exposed and not exposed insects and the cross-talk among plant pathogens, arbovirus and endosymbiont bacteria will be certainly very interesting to explore. In the very distant, but very well studied, case of honeybee colony collapse disorder induced by Varroa destructor and the associated deformed wing virus (DWV), it is worth to know that, in the absence of the parasitic mite V. destructor, DWV infection is asymptomatic and no apparent negative impact on honeybee fitness is reported [4]. A mutualistic symbiosis between V. destructor and DWV has been recently demonstrated [5]: the mite acts as vector of the viral pathogen, whereas DWV modulates the honeybee humoral immune response, facilitating mite feeding and reproduction.
How can vector-associated viruses affect insect behavior, plant pathogen transmission efficiency and, ultimately, plant response to infection when they are co-transmitted with plant pathogens to the plant? This is really a fascinating and largely unexplored field of investigation.

[1] Marcone C., 2014. Molecular Biology and pathogenicity of phytoplasmas. Annals of Applied Biology 165: 199-221
[2] Rashidi M., D’Amelio R., Galetto L., Marzachì C., & Bosco D. 2014. Interactive transmission of two phytoplasmas by the vector insect. Annals of Applied Biology 165: 404-413
[3] Van Oers M. M., 2010. Genomics and biology of Iflaviruses. Insect Virology Caister Academic Press, Norfolk, 231-250
[4] de Miranda J. R. & Genersch E., 2010. Deformed wing virus. Journal of Invertebrate Pathology 103: Supplement, S48-S61
[5] Di Prisco G., Annoscia D., Margiotta M., Ferrara R., Varricchio P., Zanni V., Caprio E., Nazzi F. & Pennacchio F., 2016. A mutualistic symbiosis between a parasitic mite and a pathogenic virus undermines honey bee immunity and health. PNAS 113: 3203-3208

On Enhancing Diversity in STEM, part 2

Way back in December of last year I wrote a post about developing an ethic for enhancing diversity in the STEM fields. In it, I focused on one dimension of the case for enhancing diversity among scientists: that post-positivism in science provides a coherent epistemology and ethic on which to ground diversity work.

Yet there remain a number of other dimensions of the case for diversity in STEM. One more pragmatic is the argument that diversity improves the ability of scientists to solve problems within their research projects. Diverse people bring their different experiences, areas of expertise, and ways of thinking to bear on a set of problems, and this can bring about novel solutions. This idea is convincingly discussed in a recent episode of the highly entertaining podcast Reply All. Although the discussion focuses on the problem of diversity in Silicon Valley tech companies (where the workforce may be even more homogeneous than in many STEM fields), the insights into how diversity can improve problem solving and productivity are highly relevant to academic STEM disciplines as well.

(Note, the first half of the podcast episode is about an obscure area of the “Twitter-sphere”. It’s pretty funny and entertaining but not particularly relevant; skip to the second half if you’re in a hurry. Also note: ironically, the hosts of the podcast completely ignored Rosalind Franklin’s contribution when they touched on the discovery of the structure of DNA. They have a follow-up episode where they atone.)

The story in the podcast made me think about the dynamics in the Almeida lab. We are a disciplinarily diverse lab, with expertise in molecular biology, genomics, plant pathology, entomology, macro-ecology, and modeling. We are also a multinational and multicultural lab.  It’s easy to see how diverse disciplines can improve scientific work, particularly when addressing environmental or agricultural problems. My own work has undoubtedly benefited from working with molecular and micro-biologists in the lab. While it remains less clear how other dimensions of our identities (e.g., nationality, race, class, sexual orientation) could improve our work, the ideas put forward in the Reply All podcast make a convincing case for thinking more broadly about diversity. At the very least, it’s a worthwhile question to consider and pursue.

Some question about Candidatus Liberibacter solanacearum transmission by psyllids

Candidatus Liberibacter solanacearum (CaLso) is a phloem Gram-negative limited bacterium causing the “Zebra chip” (ZC) in potato and other vegetative disorders in other Solanaceae crops (i.e. tomatoes, peppers, tamarillos) in Central, North America and New Zealand. Interestingly the bacterium has been detected in Finland, Sweden, Germany, Norway, and Canary Islands and in the mainland Spain causing disease in carrots and celery commercial crops.

CaLso is transmitted from potato mother tubers to growing plants and to progeny tubers. Moreover, CaLso is naturally spread by different species of psyllids (Hemiptera: Triozidae), a group of phloem feeders that have been always correlated with leaf yellows and yield crop reduction. While the American potato psyllid, Bactericera cockerelli, is the known vector of CaLso in solanaceous plants in North America and New Zealand, two different psyllid species have been reported so far as vectors of CaLso in Apiaceae crops: Trioza apicalis in northern Europe and Bactericera trigonica in the Mediterranean region.

Five different haplotypes of the bacterium have been described (designated in order of description as A, B, C, D and E) according to their ability to infect different crops in specific geographical regions. CaLso haplotypes A and B occur on Solanaceae plants in America and New Zealand and CaLso haplotypes C, D, E occur on members of the Apiaceae family in northern Europe and the Mediterranean regions.

It is known that CaLso is transmitted in a propagative-circulative manner (it needs to replicate in the vector body before inoculation) by vectors and that vector salivation and vector sap ingestion into the phloem sieve tubes are an absolute prerequisite for pathogen transmission.

Due to its relative recent emergence, there is a lack of information on aspects related to the transmission, vector-bacteria interactions and the epidemiology of CaLso. For example, there are few studies on the transmission characteristics of CaLso by their psyllid vectors such as B. cockerelli, B. trigonica and T. apicalis as well as other psyllid species that may also act as vectors.

For example, is there any specificity for the transmission of the different bacterial haplotypes transmission by the different vector species? Is it possible the transmission of several bacterial haplotypes by different vectors between different hosts?

It is obvious that the haplotypes present in a given area (i.e. A and B in North America) are transmitted by the psyllid species that is dominant in the region (i.e. B. cockerellii). However, this fact is not indicative that there is a vector-haplotype specificity in CaLso transmission, because there might be no other potential vectors colonizing the crop on the given region of study. What would happen if a psyllid species that feeds and colonize solanaceaus crops (eg. Russelliana solanicola) is introduced into one of the potato growing areas of Oregon State in the United States (i.e)? Until now, there is not information about bacterial-vector specificity, and in my opinion, there is still much to investigate on the relationship of CaLso-vector interactions and the epidemiology of vector-borne bacterial diseases.

It is known that in the field, phloem-restricted pathogens are transmitted efficiently only by colonizing species and, from an epidemiological point of view, the transmission of a persistent phloem-restricted plant pathogen by non-colonizing species is very unlikely to occur in the field. That fact would explain why B. cockerellii is the main vector of CaLso in potato in North America or B. trigonica is the main vector in carrots in Spain. However, this does not exclude the possibility that other psyllid species sporadically probe on a non-host crop, particularly if that crop is the only available in the field at a given time of the year. The rate of plant pathogen transmission would be lower than expected but this psyllid species could be an occasional vector to crops other than its main host. Probably this psyllid species would not be involved in the secondary spread of the pathogen but it would be responsible to its primary spread acquiring a main role on the bacterium epidemiology.

So, if the previous assumption would be possible, could a given bacterial haplotype able to infect Apiaceae crops become a serious problem in Solanaceae crops (i.e)? That would be one question that could be considered in areas where potato crops are growing close to carrot crops, for example. Would the infected carrots act as a primary inoculum source of the bacterium? Could other weeds and natural vegetation near to the crops be the potential reservoirs CaLso? Would a given psyllid species colonizing carrots (i.e. B. trigonica) be responsible of an occasional transmission to potatoes? Or is there any other psyllid species able to feed sporadically in carrots and then feed on potato transmitting the bacteria?

Obviously, the first step to solve this enigma would be to verify if the same haplotype is able to inoculate both crops (eg carrot and potatoes) . Once this is known, the second thing to do would be to identify the potential vectors of the bacterium.

Take as an example the situation in Spain (because it is the most close to me). Both crops, Solanaceae and Apiaceae, can share growing regions simultaneously for some time periods, so it would be possible that this theoretical situation occurs. The most abundant psyllid species found in carrots in Spain is B. trigonica that is the main vector of CaLso in carrots. However, B. tremblayi and B.nigricornis have been found in carrots, celery or potato crops. Until now, it is not yet clear if B. trigonica is able to transmit the bacterium to other crops or if these other psyllid species could be involved in the spread of CaLso in the field.

In any case, if this unknown psyllid species is able to feed on different crops it could become a “potential vector” of CaLso on different crops. For example, if viruliferous psyllids can fly from Apiaceae-infected hosts to solanaceous crops (or viceversa) and then land and feed for a short time they would move the primary inoculum to an unexpected host plant. This is an example of why it is mandatory to improve our knowledge on this epidemiological aspect in order to prevent or reduce the spread of the bacterial under field conditions.

Some references:

  • Alfaro-Fernández A et al., 2012. Plant Disease 96, 581.
  • Bertolini E et al., 2014. Plant Pathology 64, 276-285.
  • Irwin ME et al., 2007. Aphids as Crop Pests, pp. 153-186. In: Aphid Movement: Process and Consequences. Van Emden HF and Harrington R. (eds). CABI Publishing, Wallingford.
  • Liefting LW et al. 2009. Plant Disease 93, 208-214.
  • Munyaneza JE et al., Journal of Economic Entomology 103, 1060-1070.
  • Munyaneza JE et al., 2010. Plant Disease 94, 639.
  • Munyaneza JE, 2012. American Journal of Potato Research 89, 329-350.
  • Munyaneza JE, 2015. American Journal of Potato Research 92, 230-235.
  • Pitman AR et al., 2011. European Journal of Plant Pathology 129, 389-398.
  • Secor, GA et al., 2009. Plant Disease 93, 574-583.