Dissecting phytoplasma effector adaptation to plant targets (Bilateral BBSRC-FAPESP application)

Lead Research Organisation: John Innes Centre
Department Name: Cell and Develop Biology

Abstract

Phytoplasmas are intracellular bacterial pathogens of plants that are transmitted by insect vectors. They induce a variety of symptoms in plants and crops, such as witches' brooms (increased lateral branching) and phyllody (flowers reverting into leaves that remain sterile). Phytoplasmas can negatively impact crop production in many regions of the world. They interfere with flower production and decline fruit/grain yields, a problem in maize, fruit trees, grapevines, coconut etc. Because phytoplasmas are insect-transmitted, their occurrence is expanding as the climate warms up and pesticide use is being restricted.

The Hogenhout (SH) group at The John Innes Centre has made significant progress with phytoplasma pathogenesis and their impact on host plants. This group found that phytoplasmas produce specific virulence proteins (effectors), which interact with conserved plant proteins leading to crinkled leaves, increased lateral branching (witches' brooms) and the downregulation of plant defence responses to the phytoplasma insect vectors. The latter leads to a greater number of insect vectors that transmit the phytoplasma to other plants. So far, the SH group has focused on phytoplasma interactions with the model plant Arabidopsis thaliana.

In this project, the SH group will collaborate with the Spotti-Lopes (JSL) group at The University of Sao Paulo in Brazil who has studied the ecology and epidemiology of an important phytoplasma disease agent of maize. This pathogen causes maize bushy stunt disease and is transmitted by the corn leafhopper, which builds up to high population levels in maize fields in Brazil, Argentina and Mexico, therefore triggering severe phytoplasma epidemics. Maize bushy stunt phytoplasma and the corn leafhopper have co-evolved with the domestication of maize from its wild progenitor, teosinte, and hence are well adapted to colonize maize. Phytoplasma-infected maize plants typically produce more primary and secondary lateral branches (hence, the name maize bushy stunt phytoplasma), fewer ears and lower grain yields. These infected plants may support a greater number of corn leafhoppers that transmit maize bushy stunt phytoplasma to other maize plants.

Together the SH and JSL group will translate the knowledge from the phytoplasma-Arabidopsis system to investigate maize bushy stunt phytoplasma and the corn leafhopper in maize. More specifically, we will determine if a maize bushy stunt phytoplasma effector protein induces the increased branching and reduced grain yield symptoms typically observed in phytoplasma-infected maize. Moreover, we will determine of this effector suppresses plant immunity to the corn leafhopper leading to greater leafhopper populations. We will also investigate the genomic variation of maize bushy stunt phytoplasmas in Brazil. Finally, we will assess if we can generate maize varieties that are not targeted by the phytoplasma effector protein leading to a reduction in maize bushy stunt symptoms and increased grain production during phytoplasma epidemics. We will collaborate with the agricultural company Dow Agrosciences. This project will provide exchange visits and a learning platform for all staff involved. Overall this work will increase our fundamental understanding of how pathogen virulence factors (effectors) adapt to host targets thereby facilitating pathogen colonization of these hosts and pathogen dispersal by insect vectors.

Technical Summary

Phytoplasmas are intracellular bacterial pathogens of plants that are transmitted by insect vectors, such as leafhoppers. They induce a variety of symptoms in plants and crops, such as witches' brooms (increased lateral branching) and phyllody (flowers reverting into leaves that remain sterile).

The Hogenhout group at The John Innes Centre has identified the virulence protein (effector) SAP11 from Aster Yellows Witches' Broom phytoplasma (AY-WB) that destabilize class II CIN members of the TCP transcription factor family, which are conserved among plants. SAP11-mediated CIN-TCP destabilization induces crinkled leaves and reduces synthesis of the defense plant hormone jasmonic acid in Arabidopsis. This leads to an increase in the number of aster leafhoppers that transmit AY-WB to other plants. We have also evidence that SAP11 interacts with class II CYC/tb1 TCPs that include Arabidopsis Branched 1 (BRC1), possibly resulting in increased branching (witches' brooms) symptoms of AY-WB-infected plants.

The SH group has identified a homolog of the SAP11 protein (SMP11) in the genome of Maize bushy stunt phytoplasma (MBSP). SMP11 induces increased branching but not crinkled leaves in Arabidopsis suggesting that SMP11 interacts with class II CYC/tb1 TCPs, but not the CIN-TCPs. It is also possible that SMP11 interacts with maize CIN-TCPs, but not Arabidopsis CIN-TCPs.

In this project we will collaborate with the Spotti-Lopes group at The University of Sao Paulo University in Brazil who are experienced with the ecology and epidemiology of MBSP, an important phytoplasma disease agent of maize in Brazil. We will (1) investigate if SMP11 and SAP11 interact with various classes of Arabidopsis and maize TCPs, (2) study genomic variations Brazilian MBSP isolates, and (3) Investigate the contributions of SMP11 and SAP11 to phytoplasma symptom development and leafhopper-plant interactions. Overall, this work increases our understanding of pathogen-host adaptation processes.

Planned Impact

Maize can be used as food, animal feed or to make biofuels. It is the most widely grown crop in the Americas and has been introduced into many other countries worldwide. Corn is included in the focus of the European Biofuels Technology Platform to develop new technologies for industrial-scale production of Second Generation (2G) biofuels (advanced biofuels) from sustainable feedstocks, because corn stover (i.e. leaves and stalks of corn plants left in the field after harvesting the edible corn grain) could supply up to 25% of the biofuel crop needed by 2030.

Maize bushy stunt phytoplasma (MBSP) is a major pathogen associated with corn stunt disease in Brazil and throughout Latin America, which is transmitted by the corn leafhopper, Dalbulus maidis. It has a wide geographical distribution that spans much of South America, Central America and the southern United States. Maize plants diagnosed with corn stunt disease exhibit a variety of symptoms, including increased branching, development of several small ears, ears with loose kernels, and overall plant stunting. Maize fields with high incidences of this disease show severe yield losses (up to 100%), affecting the production of food for both livestock and human consumption. The work proposed in this project will advance the use of corn as a sustainable, advanced biofuel (second generation and beyond) that is a focus in the BBSRC funding priority area Crop Science.

Strategies to control phytoplasmas typically rely on the use of pesticides to manage insect vector populations, with limited success in preventing disease spread. Approximately 80% of corn grown the USA is genetically modified (mostly Bt maize hybrids) and use of Bt maize is increasing exponentially in South American countries (particularly in Brazil and Argentina). Thus, insecticide treatments that were previously used to control lepidopteran pests will now be reduced. As a result, corn stunt disease incidence is likely to increase. This work will be useful for the identification of maize lines that are more resistant to the induction of increased (lateral) branching by MBSP infection (through genetic modification, tilling or classical breeding approaches).

This project will involve the participation of Dow AgroSciences, Brazil. They will contribute in-kind to this project by coordinating the leaf sampling in three important maize growing regions that are frequently affected with MBSP infections in Brazil. Genetic variability and pathogenicity mechanisms have not yet been investigated for MBSP and this information will be of great value for breeders.

We also plan to present talks in conferences largely attended by maize growers and technical personnel, such as the 'Brazilian Congress of Maize and Sorghum' or the 'Annual Seminar of "Safrinha" (second season) Maize'.

This work fits with the BBSRC themes "research on insect pests that affect crop performance", "translation of research outputs into practical use and application by the agriculture and food industries" and "alternatives to chemical inputs" that are listed under the BBSRC funding priority area Crop Science. It also adheres to "pests and diseases of crops and livestock" listed under the BBSRC funding priority area Living with Environmental Change. This work also takes a systems approach by combining molecular and ecological aspects of bacterial adaptation to plants and broadly fits with the BBSRC theme "Systems based approach to biology". This project seeks support from multiple agencies (BBSRC and FAPESP). Moreover it translates fundamental research obtained from a model system to and agriculturally relevant pathosystem. Thus, this project contributes to BBSRC's mission to promote interdisciplinary and whole systems approaches to research on UK and global food supplies.
 
Title Produced a video 
Description Generated a video explaining how plants become zombies and posted this on YouTube. The videos features researchers explaining the research and doing experiments, and plants with disease symptoms and insect pests. 
Type Of Art Film/Video/Animation 
Year Produced 2014 
Impact The video was released as part of a press release for the MacLean et al., 2014. PLoS Biol publication. It received attention from journalists worldwide who covered the findings in newspapers, local journals and prestigious news letters to engage the general public. 
URL https://www.youtube.com/watch?v=QGTESwEeqsM&feature=youtu.be
 
Description In this project, we made use of the expertise of the SH and JSL groups and exploited our knowledge of SAP11-TCP interactions to translate research and investigate the mode of action and epidemiological implications of effectors associated with maize bushy stunt disease.

Our long-term goal is to understand how microbial pathogens and insect pests modulate host responses to aid their colonization of plant hosts and dispersal in nature.

The goal of this project was to investigate the contributions of SAP11 effectors to AY-WB and MBSP virulence.

Arabidopsis and maize TCPs were cloned and tested for interactions with SAP11 homologues of various phytoplasmas. Chimeras of SAP11s were synthesized and examined for interactions with TCPs. As well, chimeras of the TCP domains of TCP transcription factors were examined for binding to SAP11. Single amino acid mutants for some TCPs were generated and analyzed for (loss of) binding to SAP11. We generated single and double A. thaliana mutants and miRNA-based knock down lines for various TCPs and analyzed these for developmental phenotypes and susceptibility to insects. Transgenic lines for SAP11 homologues of various phytoplasmas were generated and analyzed for developmental and insect-susceptibility phenotypes.

We showed that the phytoplasma SAP11-mediated break down of TCPs differentially impact the dicot Arabidopsis thaliana and the monocot maize (Zea mays L.). In both Arabidopsis and maize, the SAP11 break down of TCPs induces witch's broom-like shoot proliferations and changes in leaf development. However, only in Arabidopsis the SAP11 break down of some class II TCPs also suppresses defence responses of leaves to insect vectors on which phytoplasma depend for spread. Moreover, SAP11 induces more severe architectural changes in maize, including sterility and conversion of female and male organs. This work demonstrates that phytoplasma SAP11 effector targeting of class II TCP transcription factors has different outcomes in divergent plant species that, in turn, is likely to shape effector evolution depending on the predominant plant host ranges of these adaptable parasites. The work was submitted for publication and is currently under review.

We also collected MBSP isolates throughout Brazil and maintained these in the greenhouse facilities of ESALQ (JRS lab) via insect transmission. The symptoms of 5 MBSP isolates were analyzed on several different maize lines. Infected maize lines were also examined for insect vector attraction and susceptibility. The SAP11 genes of 7 MBSP isolates were amplified and sequenced and the presence of surrounding genes on the SAP11 PMU-like sequence were examined via PCRs. One MBSP genome was sequenced to completion and good quality draft genome sequences of five additional MBSP isolates were obtained.

We found that MBSP isolates contribute consistently to organ proliferation symptoms and maize genotype to leaf necrosis, reddening and yellowing of infected maize plants. The symptom differences are associated with polymorphisms in a phase-variable lipoprotein, which is a candidate effector, and an ATP-dependent lipoprotein ABC export protein, whereas no polymorphisms were observed in other candidate effector genes. The work has been published (Orlovskis et al., 2016. Annals of Botany).

The project contributed to the PhD projects of two students.
Exploitation Route The generated information has provided more knowledge about the genomic diversity of MBSP isolates in South and Central America that is useful for identifying maize lines that are more resistant to MBSP. This information will help to increase maize yields under maize bushy stunt disease occurrence. The results were communicated to maize breeding companies in Brazil who participated in the project. It has also improved our understanding of what TCP transcription factors regulate in the monocot maize.
Sectors Agriculture, Food and Drink,Chemicals,Education,Environment

URL https://www.jic.ac.uk/phytoplasma/
 
Description We engaged with maize-breeding companies in Brazil who are using the information generated in this project. We also engaged with scientists in Oman who have used the knowledge generated in this project to better understand how a phytoplasma pathogen destroys lime trees all over Oman and in the Middle East. The project also helped to promote the careers of several scientists, including a PhD student who is now a postdoctoral researcher at a University in Switserland and a Brazilian visiting postdoctoral researcher who obtained a permanent position at a state research institute in Brazil. The project provided preliminary data for proposals, which were funded by a prestigious Human Frontier Science Program Research grant and a PhD studentship. The results generated in this project have increased knowledge on how TCP transcription factors regulate development of female and male organs and corn production of maize and how the functions of TCPs in maize compare to those of Arabidopsis. It has also become apparent that TCPs regulate defense responses to insects in Arabidopsis leaves, but not in maize leaves. The work has been published.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Education,Environment
Impact Types Cultural,Societal,Economic

 
Description Human Frontier Science Program (HFSP) Research Grant
Amount $1,350,000 (USD)
Funding ID RGP0024/2015 
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 08/2015 
End 07/2019
 
Description The Royal Society Kohn Fellowship awarded to Adi Kliot
Amount £65,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 10/2018
 
Description Formal research collaboration with Abdullah Al-Sadi, Sultan Qaboos University, Muscat, Oman. 
Organisation Sultan Qaboos University
Department Department of Crop Sciences
Country Oman 
Sector Academic/University 
PI Contribution Provided intellectual input into investigating an important pathogen of lime trees in Oman, and hosted Ali Al-Subhi, PhD student at Sultan Qaboos University, to conduct molecular research on the pathogen for 6 months in 2015/2016 and 3 months in 2017.
Collaborator Contribution Provided funding for research conducted by the visiting PhD student and hosted me to visit phytoplasma-infected lime orchards and vegetable farms in Oman.
Impact Co-authored scientific publications, including Al-Subhi et al., 2017. BMC Microbiol. 17: 221.
Start Year 2014
 
Description Formal research collaboration with Chih-Horng Kuo, Academia Sinica, Taipei, Taiwan 
Organisation Academia Sinica
Department Institute of Plant and Microbial Biology
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution I provided intellectual input into the development of the research question, approaches and methodology. My team provided knowledge about the biology of the organisms, collected biological material, extracted DNA and generated raw reads of genome sequence data.
Collaborator Contribution The partner assembled the genome reads into contigs and annotated the genomes.
Impact Several co-authored publications, for example: Orlovskis et al., 2017. Ann Bot. 119: 869-884.
Start Year 2014
 
Description Formal research collaboration with Henryk Pospieszny, Institute of Plant Protection, Poznan, Poland 
Organisation Institute of Plant Protection, National Research Institute
Country Poland 
Sector Public 
PI Contribution My lab hosted Agnieszka Zwolinska, a PhD student at the Institute of Plant Protection, Oct 2016 - Mar 2017. Agnieszka conducted work on the HFSP collaborative project.
Collaborator Contribution The team in Poznan collects plant and insect samples in northern and southern regions of Poland for the HFSP project; the samples are assayed for the presence of phytoplasma strains and effector genes.
Impact We wrote a HFSP project proposal together that was funded. We hold regular Skype meetings (in average, once in the 6 weeks) to discuss progress and results.
Start Year 2014
 
Description Formal research collaboration with Joao Spotti-Lopes, University of Sao Paolo - College of Agriculture (ESALQ) 
Organisation University of Sao Paulo
Department College of Agriculture
Country Brazil 
Sector Academic/University 
PI Contribution Provided intellectual input into development of a collaborative research proposal, expertise in specific research methodologies and trained staff in molecular biology methods. Members of the partner team visited my team to gain experience with molecular biology techniques and discuss project progress. These included: - Visit of JSL to Hogenhout lab, 11-18 Sep. 2013. - Visit of JSL to Hogenhout lab, 20-24 Oct. 2014. - Visit of postdoctoral researcher Maria Cristina Canale from JSL lab, March - Dec, 2014.
Collaborator Contribution Provided intellectual input into development of a collaborative research proposal, expertise in specific research methodologies and trained and hosted staff for conducting field-based experiments. Members of my team visited the partner lab for field trips and collection of MBSP isolates and leafhoppers from infected maize fields. These included: - Field trip and processing of samples in Brazil of a PhD student in my team, 18 May - 8 June, 2013. - Visit of Hogenhout to partner lab, 28-31 Oct, 2013. - Field trip and processing of samples in Brazil, 6-21 June, 2014. - Visit and processing of samples in Brazil, 19-23 Oct, 2015.
Impact We published several papers together, e.g. Orlovskis et al., 2017. Ann. Bot. 119: 869-884, and are working on a few other manuscripts.
Start Year 2013
 
Description Formal research collaboration with Martina Seruga-Music, University of Zagreb, Croatia 
Organisation University of Zagreb
Department Department of Molecular Biology
Country Croatia 
Sector Academic/University 
PI Contribution Provided expertise on genome-wide analyses and identification and functional characterization of effectors of phytoplasmas. Hosted members of the Croatia team several times and helped the team to obtain a research grant in Croatia.
Collaborator Contribution The Croatia team provided useful sequence information to my team and contributed to the writing of publications.
Impact Exchanged useful data and co-published scientific publications. Another publication is in preparation and will be submitted in March 2018.
Start Year 2010
 
Description Formal research collaboration with Richard Immink, Wageningen University and Research Centre, The Netherlands 
Organisation University of Wageningen
Department Department of Plant Sciences
Country New Zealand 
Sector Academic/University 
PI Contribution We provided knowledge on phytoplasma virulence proteins that interfere with plant development and involved the Wageningen team into a interdisciplinary project proposal that received funding from HFSP.
Collaborator Contribution The Wageningen team provided expertise on plant transcription factors targeted by phytoplasma effectors and helped us with interpretation of the plant developmental phenotypes. They are co-investigators on our HFSP grant.
Impact We received funding for a HFSP research project. We co-published a paper: MacLean et al., 2014. PLoS Biol. 12(4):e1001835. We co-supervise postdoctoral researchers. We hold regular Skype meetings (in average once per 6 months) for the past 3 years.
Start Year 2014
 
Description Formal research collaboration with Russel Groves, University of Wisconsin, Madison, USA 
Organisation University of Wisconsin-Madison
Department Department of Entomology
Country United States 
Sector Academic/University 
PI Contribution We involved the University of Wisconsin team into a HFSP research proposal that got funded.
Collaborator Contribution The University of Wisconsin team collects phytoplasma-infected plant and leafhopper samples from all over the USA for the HFSP-funded project.
Impact We received funding for collaborative HFSP project. We hold regular progress meeting (in average once/6 weeks). We co-supervise postdoctoral researchers and PhD students. We are writing up a manuscript.
Start Year 2014
 
Description AMM at APS, Minneapolis, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A postdoc presented an invited talk in teh session Interconnected lifecycles: multitrophic interactions between plants, pathogens and insects at the American Phytopathological Society (APS) - Canadian Phytopathological Society Joint Meeting, Minneapolis, USA, 9-13 August 2014. The talk sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2014
 
Description Annual Symposium Lausanne, Switserland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a seminar in the Annual Symposium Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland, 7 Nov 2014. This sparked questions and discussion afterwards. I engaged with leading and young scientists to discuss research.
Year(s) Of Engagement Activity 2014
 
Description Biotechnology conference, Cuba 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a talk in a parallel session at the Biotechnology Conference, Havana, Cuba, 1-5 Dec 2014. This sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2014
 
Description COST meeting, Copenhagen, Denmark 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I have a talk at the Plant-Insect-Microbe Interactions Symposium, Copenhagen, Denmark, 16 April 2015. This sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2015
 
Description Chaired session at IPMB, Iguassu Falls, Argentina 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organized and chaired a parallel session on plant-insect interactions at the International Plant Molecular Biology 2015 meeting, Iguassu Falls, Argentina - Brazil, 25-30 October 2015. Established contacts with leaders in the field and engaged with students and postdoctoral researchers.
Year(s) Of Engagement Activity 2015
 
Description Co-chaired session at MPMI meeting, Rhodes, Greece 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Co-chaired concurrent session Neglected Systems at the International Congress of Molecular Plant-Microbe Interactions (MPMI), Rhodes, Greece, 6-10 July 2014. I established contacts with leaders in the MPMI field and engaged with students and postdoctoral scientists.
Year(s) Of Engagement Activity 2014
 
Description Co-chaired session at SEB, Manchester, UK 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I organized a co-chaired the session Mechanisms of Plant-Insect Interactions at the Society of Experimental Biology (SEB) meeting, Manchester, UK, 1-5 July 2014. I invited colleagues who are leaders in the field of Plant-Insect Interactions from academic institutions worldwide to present talks in this session. A student and a postdoc in my team presented talks as well and engaged with the international speakers and the audience.
Year(s) Of Engagement Activity 2014
 
Description EPS, Soest 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I presented a talk at the Dutch Graduate School Experimental Plant Sciences (EPS), Soest, The Netherlands (invited by PhD students), 29-30 Jan 2015. The audience was predominantly postgraduate students.
Year(s) Of Engagement Activity 2015
 
Description MCC at SEB, Manchester, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A visiting postdoc in the lab presented a talk in the parallel session Mechanisms of Plant-Insect Interactions at the Society of Experimental Biology (SEB) meeting, Manchester, UK, 1-5 July 2014
Year(s) Of Engagement Activity 2014
 
Description OARDC, Wooster, OH, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented a talk at my former workplace: The Ohio State University - Ohio Agriculture and Development Center (OARDC), Wooster, Ohio, USA. 10-13 May 2015. This sparked questions and discussions afterwards.
Year(s) Of Engagement Activity 2015
 
Description Phytopathological Society, Colmar, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented a talk at the French Phytopathology Meeting, Colmar, France, 2-5 June 2015. This sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2015
 
Description Phytopathology conference, Brazil 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact SH presented a talk at the 46th Meeting of the Brazilian Phytopathological Society/11th Brazilian Meeting on Biological Control, Ouro Preto, Brazil, 20-25 October 2013.
Year(s) Of Engagement Activity 2013