APPLICATION FORM Containment – GMO Project

To obtain approval for projects to develop genetically modified organisms in containment

Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.

Application Number

APP203205

Date

02/10/2017

www.epa.govt.nz 2

Application Form Approval for projects to develop genetically modified organisms in containment Completing this application form

1. This form has been approved under section 42A of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers projects for development (production, fermentation or regeneration) of genetically modified organisms in containment. This application form may be used to seek approvals for a range of new organisms, if the organisms are part of a defined project and meet the criteria for low risk modifications. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 2. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise. Commercially sensitive information

10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available.

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Application Form Approval for projects to develop genetically modified organisms in containment

12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions

Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation

Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment

Any organism in which any of the genes or other genetic material: • Have been modified by in vitro techniques, or Genetically Modified • Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques

A new organism is an organism that is any of the following: • An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998; • An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation; • An organism for which a containment approval has been given under the HSNO Act; • An organism for which a conditional release approval has been given under the HSNO Act; New Organism • A qualifying organism approved for release with controls under the HSNO Act; • A genetically modified organism; • An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand; • An organism present in New Zealand before 29 July 1998 in contravention of the Animals Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because: • It is subject to a conditional release approval; or • It is a qualifying organism approved for release with controls; or • It is an incidentally imported new organism

An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal

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Application Form Approval for projects to develop genetically modified organisms in containment 1. Applicant details

1.1. Applicant

Company Name: (if applicable) University of Canterbury

Contact Name: Mitja Remus-Emsermann

Job Title: Lecturer Microbiology

Physical Address: Kirkwood Avenue, Ilam, Christchurch

Postal Address (provide only if not the same as the physical): Private bag 4800, Christchurch

Phone (office and/or mobile): 027 459 7338 or 0 (3) 36 95351

Fax:

Email: [email protected]

1.2. New Zealand agent or consultant (if applicable)

Company Name:

Contact Name:

Job Title:

Physical Address:

Postal Address (provide only if not the same as the physical):

Phone (office and/or mobile):

Fax:

Email:

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Application Form Approval for projects to develop genetically modified organisms in containment

2. Information about the application

2.1. Brief application description Approximately 30 words about what you are applying to do

The project aims to characterise plant colonising bacteria and to understand how bacterial communities, including plant and human pathogens, but mainly non-pathogenic bacteria, colonise plant leaves.

2.2. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it

Individual plant leaves are colonised by often more than 1,000,000 bacteria that may be very diverse and are mostly not harmful to the plant or a consumer of that plant. We are aiming to understand how those bacteria, and relevant plant and human pathogens, are able to form communities on leaves, and infect the leaf respectively. To be able to do so, we plan to genetically modify plant leaf colonising bacteria, as well as model bacteria for plant disease and faecal contamination of crops.

The bacteria will be modified using genes that produce proteins that fluoresce (e.g. GFP) and which are commonly used in bacteria, fungi, plants and animal genetic engineering. These proteins make no contribution to the ability of the organism to cause disease or environmental harm. The purpose is to cause the modified organism to ‘glow’ and this permits us to visually track the organism during the experiment. This will allow their investigation on leaves using microscopy. In addition, we will knockout, i.e. destroy, genes or add additional genes to better understand their functions in plant leaf colonising bacteria.

A better understanding of plant leaf colonisation by microorganisms is important for our fundamental understanding of the ecology of plants and their bacterial colonisers. Moreover, our work will help to develop tools for future biological control applications of pathogens. This explicitly includes pathogens of economic importance in a NZ context such as the bacterial kiwifruit vine disease and bacterial fire blight of apples and pears. Furthermore, by gaining a systematic understanding of how bacteria colonise plants, it will be possible to better predict their behaviour on a macroscale.

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Application Form Approval for projects to develop genetically modified organisms in containment

2.3. Technical description

Briefly describe the host organism(s) and the proposed genetic modifications. Please make sure that any technical words used are included in a glossary. Note if any part of this research project is already covered by an existing HSNO Act approval that your organisation holds or uses.

Recently a large bacterial strain library was isolated from healthy plants in Switzerland and Germany by the applicant and others (Bai et al. 2015). The library includes representatives of a large proportion of the bacterial diversity on plant leaves. The bacteria of this library were categorised by the German cell culture to be risk category 1. We aim to exploit this library, and other sources for plant bacterial isolates categorised as PC1 or PC2 originating from healthy plants or soil (a full list of species we want to develop can be found in Table 2), model pathogens (all categorised as PC2, a full list species we want to develop can be found in Table 3), and genetically modify the bacteria using different techniques and for different purposes:

• Strains will be equipped with fluorescent protein reporter genes, either

o stably inserted into their chromosomes by means of homologue recombination, transposon delivery (also see below), or CRISPR/Cas9 technology

o or on plasmid vectors

• Strains will be screened for genes important for plant-microbe, microbe-environment and microbe- microbe interactions by generating libraries of strains with random gene knockouts using transposon mutagenesis techniques based on the transposons Tn5, Tn10, Himar or Mariner

o Transposon bearing vectors will be "mobilisable" but not self-conjugative, meaning that they can only be conjugated if a helper plasmid is present or if the host contains a chromosomally integrated transfer operon. Furthermore, transposable elements will not contain an active transposase. Thereby, the transposon does not further propagate to other cells.

• Strains will be equipped with plasmid or chromosomal gain of function constructs as described above

• The strains will be equipped with conjugative plasmids to track the dynamics of horizontal gene transfer on plants

o Conjugative plasmids are not being used for cloning experiments, but to investigate the rate of conjugation. No pathogenic bacterial DNA will be cloned into these plasmids on purpose. We are planning to work with plasmids RP4 (IncP-1alpha, see Watson 1976), pKJK5 (IncPromA, Klumper 2015), pIPO2 (IncP-1epsilon, Klumper 2015), pRK2013 (Ditta 1980) and pRK600 (Finan 1986), both IncP-1, both very similar to RP4. We will perform conjugation experiments also including model pathogen strains.

o To give an assessment of the probability of a transfer of chromosomal genetic material: The plasmid RP4 is known to be able to cause HFR, however, the rates of HFR are ~ 5x10^-8, which, in our experiments is far below the limit of detection (Watson 1976) and thereby negligible. Any risk of escape will be further mitigated by us not amplifying resulting

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Application Form Approval for projects to develop genetically modified organisms in containment transconjugants, but merely counting colonies before they are being destroyed by autoclaving. An accidental escape, or infection of the experimenter is extremely unlikely.

Unmodified risk group 1 bacteria will be imported under permit NOC100168 and genetically modified bacteria developed elsewhere under permit GMC100216. Unmodified risk group 2 bacteria will be imported under permit NOC100169 and risk group 2 bacteria developed elsewhere will be imported under permit GMC100219. Plant model species (Table 1) will cultivated under PC2 conditions (see also proposed containment section) and will be inoculated with bacteria (Table 2 and 3) under PC2 conditions. All plants and tissue cultures save Arabidopsis thaliana will be sourced from New Zealand sources such as research institutes, universities or garden centers. A. thaliana wild types will be imported under permit and genetically modified A. thaliana developed elsewhere will be imported under permit GMC100230 (e.g. the Arabidopsis Biological Resource Center (ABRC), the Nottingham Arabidopsis Stock Center (NASC), the RIKEN Bioresource Center (BRC)/ SENDAI Arabidopsis Seed Stock Center (SASSC), the INRA-Versailles Genomic Resource Center, or Lehle Seeds).

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Application Form Approval for projects to develop genetically modified organisms in containment Table 1 Plant species used in this project. Plants will include wildtype plants and genetically engineered Arabidopsis thaliana developed elsewhere

Organism Common name Phylogeny (based on NCBI taxonomy browser) Category Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Arabidopsis thaliana L. Thale cress B Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; malvids; Brassicales; Brassicaceae; Camelineae Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Solanum lycopersicum Tomato B Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; asterids; lamiids; Solanales; Solanaceae; Solanoideae; Solaneae Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Solanum tuberosum L. Potato B Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; asterids; lamiids; Solanales; Solanaceae; Solanoideae; Solaneae Actinidia deliciosa Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Actinidia chinensis Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Kiwifruit B Actinidia argute Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; asterids; Actinidia eriantha Ericales; Actinidiaceae Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Malus domestica Borkh Apple B Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; fabids; Rosales; Rosaceae; Maloideae; Maleae Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Vitis vinifera L. Grape vine B Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; rosids incertae sedis; Vitales; Vitaceae

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Application Form Approval for projects to develop genetically modified organisms in containment

3. Information about the new organism(s)

3.1. Identity of the host organism(s)

For each host organism: • Provide its taxonomic name and describe what type of organism it is. • Provide a description of the strain(s) being applied for (if relevant). • If the host organism is derived from humans (e.g., cell lines) or may have cultural significance (e.g. sourced from native biota), provide details of its source. • State the category (Category 1 or Category 2) of the host organism (as per the HSNO (Low Risk Genetic Modification) Regulations).

Table 2 describes the phylogenetic identity and origin of non-pathogenic bacterial species that will be used in the project. All organisms originate from healthy leaf material of environmentally grown Arabidopsis thaliana (Bai et al. 2015, Kämpfer et al. 2016), exceptions are the following organisms that were isolated from other healthy plant leaves or soil: 1) Sphingomonas phyllosphaere FA2 isolated from Acacia caven leaves; 2) Pseudomonas citronellolis P3B5 isolated from Ocimum basilicum; 3) Pseudomonas koreensis P19E3 isolated from Origanum vulgare; 4) Pseudomonas orientalis F9 isolated from Malus domestica; 5) Pseudomonas putida KT2440 isolated from soil; 6) Pseudomonas putida WCS358 was isolated from Solanum tuberosum tubers; 7) Pseudomonas protegens CHAO was isolated from tobacco black rot suppressive soil; and 8) Pseudomonas fluorescence PF0-1 was isolated from soil. Table 3 describes the phylogenetic identity and origin of pathogenic bacterial species that will be used in the project. Table 4 describes bacterial strains that will mainly be used for the development of genetic vectors, i.e. E. coli laboratory strains.

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Application Form Approval for projects to develop genetically modified organisms in containment

Table 2 non-pathogenic bacteria originating from healthy plant material Organism Species Strain(s) Ref Phylogeny/ notes (based on the List of Prokaryotic Names Category# with Standing nomenclature and Bergey’s Manual of Systematics of Archaea and Bacteria) Williamsia sp. DSM102764 Kämpfer et al. Actinobacteria / Actinobacteria / Corynebacteriales / Nocardiaceae 1 (1999) herbipolensis ARP1 Kämpfer et al. 1 (2016) Pantoea agglomerans 299R Remus- Proteobacteria / Gammaproteobacteria / Enterobacteriales / 2 Emsermann et al. Enterobacteriaceae (2013),

Pedobacter sp. DSM102678 Steyn et al. Bacteroidetes / Sphingobacteriia / Sphingobacteriales / 1 (1998) Sphingobacteriaceae Variovorax sp. DSM102763 Willems et al. Proteobacteria / Betaproteobacteria / Burkholderiales / 1 (1991) Comamonadaceae Pseudomonas Migula (1894) Proteobacteria / Gammaproteobacteria / Pseudomonadales / Pseudomonadaceae citronellolis P3B5 Remus- 2 Emsermann et al. 2016 koreensis P19E3 Schmid et al. in 2 prep. orientalis F9 Schmid et al. in 2 prep. putida Kt2440 Bagdasarian et al. 2 (1981) putida WCS358 Geels and 2 Schippers (1983) protegens CHA0 Jousset et al. 2 (2014) fluorescens PF0-1 Compeau et al. 2 (1988) Bradyrhizobium sp. DSM102579 Jordan (1982) Proteobacteria / Alphaproteobacteria / Rhizobiales / 1 Bradyrhizobiaceae Methylobacterium spp. DSM102621; Patt (1976) Proteobacteria / Alphaproteobacteria / Rhizobiales / 1

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Application Form Approval for projects to develop genetically modified organisms in containment DSM102634 Methylobacteriacea radiotolerans 0-1 Ito and Iizuka 1 (1971) extorquens PA1 Knief et al. (2010) 1 Sphingomonas spp. DSM102725; Yabuuchi et al. Proteobacteria / Alphaproteobacteria / Sphingomonadales / 2 DSM102734; (1990) Sphingomonadaceae DSM102732 Category based on Sphingomonas paucimobilis melonis FR1 Innerebner et al. 1 (2011) phyllosphaerae FA2 Rivas et al. (2004) 1 Methylophilus spp. DSM102653; Jenkins et al. Proteobacteria / Betaproteobacteria / Methylophilales / 1 DSM102655 (1987) Methylophilaceae Rhodococcus sp. DSM102719 Zopf (1891) Actinobacteria / Actinobacteria / Corynebacteriales / Nocardiaceae 2 Category based on Rhodococcus equi Arthrobacter sp. DSM102322 Conn and Actinobacteria / Actinobacteria / Micrococcales / Micrococcaceae 1 Dimmick (1947) Microbacterium sp. DSM102661; Orla-Jensen Actinobacteria / Actinobacteria / Micrococcales / 1 DSM102662 (1919) Microbacteriaceae Aeromicrobium sp. DSM102280 Miller et al. (1991) Actinobacteria / Actinobacteria / Propionibacteriales / 1 Nocardioidaceae Acidovorax sp. DSM102561 Willems et al. Proteobacteria / Betaproteobacteria / Burkholderiales / 2 (1990) Comamonadaceae Agreia sp. DSM102279 Evtushenko et al. Actinobacteria / Actinobacteria / Micrococcales / 1 (2001) Microbacteriaceae Rathayibacter sp. DSM102695 Zgurskaya et al. Actinobacteria / Actinobacteria / Micrococcales / 1 (1993) Microbacteriaceae Plantibacter sp. DSM102681 Behrendt et al. Actinobacteria / Actinobacteria / Micrococcales / 1 (2002) Microbacteriaceae All organisms listed above were isolated from healthy plant leaf material. The species listed above can best be described as “saprophytes” or “commensals” and potential “plant-beneficial” bacteria, their actual roles and behaviour on plants is yet to be determined. *The origin of most isolates is described in Bai et al. 2015 Nature and the strains were or will be full genome sequenced (see https://www.dsmz.de/catalogues/catalogue-microorganisms/groups-of-organisms- and-their-applications/arabidopsis.html). #Category as per HSNO low risk Genetic modification regulation.

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Application Form Approval for projects to develop genetically modified organisms in containment

Table 3 Selected attenuated human pathogenic bacteria and plant-pathogenic bacteria Organism Species Strain Ref Phylogeny/ notes Category# Escherichia coli NCTC Migula Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae 2 12900; 1895 This is a Eschericha coli O157:H7 that lacks the stx1 and stx2 genes and is not O157:H7 producing verotoxin. It is commonly used as a quality control strain for E. coli stx- serotype O157:H7 detection procedures. Salmonella enterica sv. X3895 Curtiss Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae 2 Typhimurium and Kelly This is an attenuated strain lacking adenylate cyclase and cyclic AMP receptor (1987) protein rendering it avirulent and it has previously utilized as surrogate in other model systems. Erwinia amylovora undefined Vanneste Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae 2 (2000) The causal agent of fire blight in pome fruit. Xanthomonas campestris LMG568 Pammel Proteobacteria / Gammaproteobacteria / Xanthomonadales / Xanthomonadaceae 2 pv. (1895) A model plant-pathogen for brassica, e.g. Arabidopsis thaliana. Isolated from campestris Brussel sprouts. Pseudomonas syringae pv. B728a Feil et al. Proteobacteria / Gammaproteobacteria / Pseudomonadales / Pseudomonadaceae 2 syringae (2005) a model plant-pathogen for bean plants. Isolated from diseases bush bean leaf syringae pv. DC3000 Buell et a model plant-pathogen for tomato and Arabidopsis thaliana. Isolated from diseased 2 tomato al. (2003) tomato leaf. syringae pv. undefined Takikawa important plant-pathogenic strains relevant to NZ agriculture and kiwifruit production 2 actinidiae et al. world-wide (1989) All organisms listed above are specific model strains for laboratory plant infection studies or pathogen survival studies with the exception of Erwinia amylovora and P. syringae pv. actinidiae, for which model strains and future New Zealand isolates that are of interest for local stakeholders might be used. #Category as per HSNO low risk Genetic modification regulation.

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Application Form Approval for projects to develop genetically modified organisms in containment

Table 4 laboratory strains for cloning purposes Organism Species Strain Ref Phylogeny/ notes Category# Escherichia coli K12 and Migula Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae 1 derivates (1895) Standard laboratory strains that are used as cloning hosts such as E.coli Dh5alpha, HB101, JM109, etc. BL21 and Migula Standard laboratory strains that are used as cloning hosts such as E.coli BL21, etc. 1 derivates (1895) S17-1 Migula A laboratory strain that contains the pir gene and the transfer operon of plasmids RP4 in its 1 lambda (1895) chromosome. This effectively allows the maintenance of R6K suicide plasmids and the pir conjugation of mobiliseable plasmids, respectively. #Category as per HSNO low risk Genetic modification regulation.

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Application Form Approval for projects to develop genetically modified organisms in containment

3.2. Regulatory status of the organism

Is the organism that is the subject of this application also the subject of:

An innovative medicine application as defined in section 23A of the Medicines Act 1981?

Yes No

An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?

Yes No

4. Information about the project

4.1. Describe the nature and range of the proposed genetic modifications

• Describe the nature and range of the proposed genetic modifications (e.g. the range of elements that the vectors or gene constructs may contain, and the type, source and function of the donor genetic material). • State the category (Category A or Category B) of the genetic modifications (as per the HSNO (Low Risk Genetic Modification) Regulations).

We will use vectors that contain one or more of the following genetic elements derived from bacterial, animal, fungal, plant, bacteriophages and other viruses and or of synthetic origin:

• Regulatory elements such as promoters, regulatory peptides, non-coding RNA genes, and regulatory elements for inducible expression

o Regulatory elements may be used in conjunction with fluorescent proteins to construct whole cell bioreporters that report on intracellular or extracellular conditions.

o Regulatory elements may be used to drive/ change the expression of indigenous genes or heterologously expressed genes to change the behaviour of bacterial strains on plant leaves.

• General plasmid features such as: multiple cloning sites, origins of replication, selection markers, transcriptional activators and terminators

• Antibacterial production genes

o Bacterial strains will be equipped with antibiotic production genes to test the effect of antibiosis on bacterial interactions on plants and their effect on spatial community patterns

• Reporter genes such as colorimetric, bioluminescent or fluorescent genes.

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Application Form Approval for projects to develop genetically modified organisms in containment

o In conjunction with regulatory elements, see above • Transposable elements

o The transposon bearing vectors will be "mobilisable" but not self-conjugative, meaning that they can only be conjugated if a helper plasmid is present or if the host contains a chromosomally integrated transfer operon. Furthermore, transposable elements will not contain an active transposase. Thereby, the transposon does not further propagate to other populations.

• Transposases

o Transposable elements will not contain an active transposase, the transposase will be provided in trans, on a different genetic element, or on the same element, but not in the transposable region.

• Transfer genes to conjugate self-transmissible plasmids

o Conjugative plasmids are not being used for cloning experiments, but to investigate the rate of conjugation. No pathogenic bacterial DNA will be cloned into these plasmids on purpose. We are planning to work with plasmids RP4 (IncP-1alpha, see Watson 1976), pKJK5 (IncPromA, see Klumper 2015), pIPO2 (IncP-1epsilon, see Klumper 2015), pRK2013 (Ditta 1980) and pRK600 (Finan 1986), both derived from RP4. We will perform conjugation experiments also including model pathogen strains.

o To give you an assessment of the probability of a transfer of chromosomal genetic material: The plasmid RP4 is known to be able to cause high- frequency recombination (HFR), however, the rates of HFR are ~ 5x10^-8 (Watson 1976), which, in our experiments is far below the limit of detection and thereby negligible. Any risk of escape will be further mitigated by us not amplifying resulting transconjugants, but merely counting colonies before they are being destroyed by autoclaving. An accidental escape, or infection of the experimenter is extremely unlikely.

Additional genetic material may contain one or more of the following elements:

Genes from living organisms

Single genes or operons (cDNA or genomic fragments) of bacterial origin encoding proteins that are involved in plant-bacteria or bacteria-bacteria interactions, including but not limited to phytohormone production/ degradation, secondary metabolite production, antimicrobial material production, surfactant production and non-translated RNAs. Genetic material from native New Zealand animals and plants will not be used, nor will genetic material from culturally valued species sourced from New Zealand be used.

Category A modifications of non-pathogenic bacteria

All bacteria that are mentioned above as being non-pathogenic will be developed using standard cloning and transformation techniques and will employ cloning and expression vectors from

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Application Form Approval for projects to develop genetically modified organisms in containment commercial and research laboratory sources. All laboratory work will be performed in containment under PC2 conditions.

Category B modifications of plant-pathogenic or attenuated human-pathogenic bacteria

All bacteria that are mentioned above being plant-pathogenic or attenuated human-pathogenic will be developed using standard cloning and transformation techniques and will employ cloning and expression vectors from commercial and research laboratory sources. All laboratory work will be performed in containment under PC2 conditions.

Modifications will exclude: Intentional protein expression (i.e., expression of protein from the sequences cloned into the plasmid vector), intentional production of infectious particles (excluding bacteriophage) or other modifications that intentionally increase the pathogenicity, virulence, or infectivity of the host organism or enhance its ability to escape containment are not permitted under this approval. Therefore, users of this approval must not knowingly use vector systems or donor genetic material that will produce protein or infectious particles or increase the pathogenicity, virulence, or infectivity of the host organism or enhance its ability to escape containment. Furthermore, modifications will exclude sequences derived from humans or New Zealand native flora and fauna, sequences known to produce clinically relevant antibiotic resistance, material from species covered by relevant CITES agreements, transgenes for proteins known to present a vertebrate disease risk; and genes that encode vertebrate toxins with an LD50 < 100 µg/kg.

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Application Form Approval for projects to develop genetically modified organisms in containment

4.2. Proposed containment of the new organism(s) (physical and operational)

• State which Containment Standard(s) your facility is approved to. • State the minimum containment level (PC1 or PC2 as per AS/NZS2243.3:2002) required to contain the GMOs (as per the HSNO (Low Risk Genetic Modification) Regulations). • Discuss whether controls in addition to the requirements listed in the Standard(s) are necessary to adequately contain the GMOs.

The University of Canterbury maintains MPI approved PC2 containment facilities (#559), which meet the requirements of standards:

• Containment Facility for Micro-organisms and Cell Cultures

• Transitional Facility for Biological Products

• Containment Facilities for Plants 2007

Work with the organisms will be limited to those facilities that have been MPI approved for that organism.

This includes an operational protocol and maintenance programme to prevent any release of the contained organisms and we consider the likelihood of escape to be minimal. This protocol also requires approval by MPI, and includes procedures such as the requirement of staff to wear lab-coats with no pockets and disposable 'bootees', as a precaution against the transport of pollen and seeds out of the laboratory facilities.

Plants will be grown under in vitro conditions in closed vessels on defined, sterile media before they are inoculated with bacteria under PC2 conditions and further incubated under in vitro conditions in a PC2 containment facility. This will effectively prevent the spread of pollen, seeds and bacteria so that the likelihood of escape of reproductive organs and bacteria will be minimal. Plant organs colonised with bacteria will be harvested under PC2 conditions. For microscopy, they will be transferred enclosed in at least two containers to a within house microscopy facility operating at PC2.

All transgenic material and wildtype plants inoculated with transgenic bacteria will be autoclaved prior to disposal. Consequently, there will be no impact of this research on the environment, local flora and fauna or public health.

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5. Risks, costs and benefits

Provide information of the risks, costs and benefits of the GMOs in the following areas of impact: • The environment. • Human health and safety. • The economy (e.g. the ability of people and communities to provide for their economic wellbeing). • The relationship of Māori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga, and the principles of the Treaty of Waitangi (The details of any engagement or consultations with Māori that you have undertaken in relation to this application should be discussed here). • Society and community. • New Zealand’s international obligations.

The Environment

All the organisms and their development will be in containment, and we have procedures to ensure they will not escape, therefore it is not expected that any of the organisms created will any adverse effects on the environment. The genetic modifications are all of a well described nature and can efficiently be tracked. Were an escape to occur, the organisms would be traceable both visually because of the intended modifications or using DNA techniques such as PCR.

Since most of the organisms described above were recently isolated from environmental plants one can expect that they might be able to colonise plants of the New Zealand flora too once outside of containment. They would, however, have to compete with other bacteria that naturally inhabit those plants. Therefore, although uncertain, the chain of events that would be needed to cause adverse effects is unlikely to occur raising our confidence that containment will be an adequate risk management tool before strains would be able to effectively generate viable populations in the New Zealand environment.

Also for plant pathogenic species such as some Pseudomonas spp. and Erwinia amylovora one could expect that they might be able to colonise plants of the New Zealand flora too once outside of containment. As stated above, they would have to compete with other bacteria that naturally inhabit those plants. Furthermore, the plant pathogenic strains chosen are not known to cause disease on native plant species, in some cases however, they are pathogens of agricultural crops, such as beans, kiwi fruit, cabbage, pome fruit and tomato. Although uncertain, the chain of events that would be needed to cause adverse effects is unlikely to occur raising our confidence that containment will be an adequate risk management tool before strains would be able to effectively generate viable populations in the New Zealand environment. Were an escape to occur, the full antibiotic susceptibility profile of our strains would be known.

Strains that are related to human pathogenic species such as E. coli O157:H7 stx-, Salmonella enterica sv. Typhimurium X3895 were specifically chosen because of the attenuation of pathogenic ancestral features while maintaining most of their natural behaviour and representing low risks to

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Application Form Approval for projects to develop genetically modified organisms in containment experimenter and environment. Given their attenuation, it is unlikely, however not impossible, that they might cause disease symptoms. Although uncertain, the chain of events that would be needed to cause adverse effects is unlikely to occur raising our confidence that containment will be an adequate risk management tool before the strains would be able to effectively generate an outbreak of disease in the New Zealand environment. Were an escape to occur, the full antibiotic susceptibility profile of our strains would be known and we would ensure that during development no vectors were used with genes for resistance to current agents used in response to diseases caused by these organisms.

Self-conjugative plasmids used in this project could potentially establish themselves in environmental bacterial populations and communities. Although uncertain, the chain of events that would be needed to cause adverse effects is unlikely to occur raising our confidence that containment will be an adequate risk management tool before strains would be able to effectively generate environmental populations containing self-conjugative plasmids in the New Zealand environment. Conjugative plasmids are not being used for cloning experiments, but to investigate the rate of conjugation. No pathogenic bacterial DNA will be cloned into these plasmids on purpose. We are planning to work with plasmids RP4 (IncP-1alpha, see Watson 1976), pKJK5 (IncPromA, see Klumper 2015), pIPO2 (IncP- 1epsilon, see Klumper 2015), pRK2013 (Ditta 1980) and pRK600 (Finan 1986), both RP4 derived. We will perform conjugation experiments also including model pathogen strains.

To give an assessment of the probability of a transfer of chromosomal genetic material: The plasmid RP4 is known to be able to cause HFR, however, the rates of HFR are ~ 5x10^-8, which, in our experiments is far below the limit of detection (Watson 1976) and thereby negligible. Any risk of escape will be further mitigated by us not amplifying resulting transconjugants, but merely counting colonies before they are being destroyed by autoclaving. An accidental escape, or infection of the experimenter is extremely unlikely. Were an escape to occur, the full antibiotic resistance profile conferred by the plasmids would be known and since they confer fluorescent markers, they could be easily identified by microscopy and PCR.

There are limited direct benefits for the environment as this is for research only. However, in the future the results of the planned studies will enable follow-up studies (with NZ native organisms that are non- GMOs) and development of formulations for plant-protective products using live organisms, thereby effectively reducing the amount of pesticides used in agriculture. This might lower the impact of agrochemicals on the environment. All future aspects will be subject of forthcoming license applications.

Human Health and Safety

All organisms will be in containment, and we have procedures to ensure they will not escape. Therefore, it is not expected that any of the organisms created will have adverse effects on human health. It is important to note that the human pathogens proposed for use in this permit are attenuated strains. Thereby minimising health risks for experimenter and environment.

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Application Form Approval for projects to develop genetically modified organisms in containment

The Economy

All the organisms will be in containment, and we have procedures to ensure they will not escape. Therefore, it is not expected that any of the organisms created will have adverse effects on the economy.

There are limited direct benefits to the economy as this is for research only. However, in the future the results of the planned studies might enable the formulation of plant-protective products using live organisms, thereby effectively reducing the amount of pesticides used in agriculture.

Culture

This work is not expected to impact in any way on the relationship of Maori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga.

Society and Community

All the organisms will be in containment, and we have procedures to ensure they will not escape. Therefore, it is not expected that any of the organisms created will have adverse effects on society and the community.

There are limited direct benefits for the society as this is for research only. However, in the future the results of the planned studies might enable the formulation of plant-protective products using life organisms, thereby effectively reducing the amount of pesticides used in agriculture. This might lower the impact of agrochemicals on society.

International Obligations

There are no international obligations concerning these organisms nor the modifications that we propose.

6. Other information

Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application.

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Application Form Approval for projects to develop genetically modified organisms in containment

7. Checklist This checklist is to be completed by the applicant

Application Comments/justifications

All sections of the application form completed Yes No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)

Confidential data as part of a separate, Yes No identified appendix

Supplementary optional information attached:

• Copies of additional references Yes No

• Relevant correspondence Yes No

Administration Are you an approved EPA customer? Yes No If Yes are you an:

Applicant: Agent:

If you are not an approved customer, payment Paid via purchase order of fee will be by: number PO 608826 • Direct credit made to the EPA bank Yes No account (preferred method of payment) Payment to follow Date of direct credit: 02/10/2017

• Cheque for application fee enclosed Yes No Payment to follow

Electronic, signed copy of application e-mailed Yes to the EPA

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Application Form Approval for projects to develop genetically modified organisms in containment

Signature of applicant or person authorised to sign on behalf of applicant

I am making this application, or am authorised to sign on behalf of the applicant or applicant organisation.

I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.

Signature Date

Request for information waiver under section 59 of the HSNO Act

I request for the Authority to waive any legislative information requirements (i.e. concerning the information that has been supplied in my application) that my application does not meet (tick if applicable).

Please list below which section(s) of this form are relevant to the information waiver request:

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Application Form Approval for projects to develop genetically modified organisms in containment

Appendices and referenced material (if any) and glossary (if required)

References:

Bai, Y., Muller, D. B., Srinivas, G., Garrido-Oter, R., Potthoff, E., Rott, M., . . . Schulze-Lefert, P. (2015). Functional overlap of the Arabidopsis leaf and root microbiota. Nature, 528(7582), 364-369. doi:10.1038/nature16192

Behrendt, U., Ulrich, A., Schumann, P., Naumann, D., & Suzuki, K.-i. (2002). Diversity of grass- associated Microbacteriaceae isolated from the phyllosphere and litter layer after mulching the sward; polyphasic characterization of Subtercola pratensis sp. nov., Curtobacterium herbarum sp. nov. and Plantibacter flavus gen. nov., sp. nov. International Journal of Systematic and Evolutionary Microbiology, 52(5), 1441-1454.

Buell, C. R., Joardar, V., Lindeberg, M., Selengut, J., Paulsen, I. T., Gwinn, M. L., . . . Collmer, A. (2003). The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA, 100. doi:10.1073/pnas.1731982100

Burrill, T. J. (1883). New Species of Micrococcus (Bacteria).

Compeau, G., Al-Achi, B. J., Platsouka, E., & Levy, S. (1988). Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems. Applied and Environmental Microbiology, 54(10), 2432-2438.

Conn, H., & Dimmick, I. (1947). Soil bacteria similar in morphology to Mycobacterium and Corynebacterium. Journal of Bacteriology, 54(3), 291.

Curtiss, R., & Kelly, S. M. (1987). Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infection and Immunity, 55(12), 3035- 3043.

Ditta, G., Stanfield, S., Corbin, D. & Helinski, D. R. Broad host range DNA cloning system for gram- negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77, 7347-7351 (1980).

Evtushenko, L. I., Dorofeeva, L. V., Dobrovolskaya, T. G., Streshinskaya, G. M., Subbotin, S. A., & Tiedje, J. M. (2001). Agreia bicolorata gen. nov., sp. nov., to accommodate actinobacteria isolated from narrow reed grass infected by the nematode Heteroanguina graminophila. International Journal of Systematic and Evolutionary Microbiology, 51(6), 2073-2079.

Feil, H., Feil, W. S., Chain, P., Larimer, F., DiBartolo, G., Copeland, A., . . . Lindow, S. E. (2005). Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000. Proc Natl Acad Sci USA, 102. doi:10.1073/pnas.0504930102

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Finan, T. M., Kunkel, B., De Vos, G. F. & Signer, E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. Journal of Bacteriology 167, 66-72 (1986).

Frank, B. (1889). Ueber die Pilzsymbiose der Leguminosen. Berichte der Deutschen Botanischen Gesellschaft, 7(8), 332-346. doi:10.1111/j.1438-8677.1889.tb05711.x

Geels, F., & Schippers, B. (1983). Reduction of yield depressions in high frequency potato cropping soil after seed tuber treatments with antagonistic fluorescent Pseudomonas spp. Journal of Phytopathology, 108(3-4), 207-214.

Haynes, W., & Burkholder, W. (1957). Genus I. Pseudomonas Migula 1894. Bergey’s Manual of Determinative Bacteriology, 89-152.

Horn, H., Keller, A., Hildebrandt, U., Kämpfer, P., Riederer, M., & Hentschel, U. (2016). Draft genome of the Arabidopsis thaliana phyllosphere bacterium, Williamsia sp. ARP1. Standards in Genomic Sciences, 11(1). doi:10.1186/s40793-015-0122-x

Innerebner, G. (2011). Identification of Indigenous Phyllosphere Isolates of the Genus Sphingomonas as Plant-Protective Bacteria. (PhD), ETH Zurich. (19649)

Ito, H., & Iizuka, H. (1971). Taxonomic Studies on a Radio-resistant Pseudomonas: Part XII. Studies on the Microorganisms of Cereal Grain. Agricultural and biological chemistry, 35(10), 1566-1571.

Jenkins, O., Byrom, D., & Jones, D. (1987). Methylophilus: a new genus of methanol-utilizing bacteria. International Journal of Systematic and Evolutionary Microbiology, 37(4), 446-448.

Jordan, D. (1982). NOTES: transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. International Journal of Systematic and Evolutionary Microbiology, 32(1), 136-139.

Jousset, A., Schuldes, J., Keel, C., Maurhofer, M., Daniel, R., Scheu, S., & Thuermer, A. (2014). Full- genome sequence of the plant growth-promoting bacterium Pseudomonas protegens CHA0. Genome Announcements, 2(2), e00322-00314.

Kämpfer, P., Andersson, M. A., Rainey, F. A., Kroppenstedt, R. M., & Salkinoja-Salonen, M. (1999). Williamsia muralis gen. nov., sp. nov., isolated from the indoor environment of a children's day care centre. International Journal of Systematic and Evolutionary Microbiology, 49(2), 681-687. doi:doi:10.1099/00207713-49-2-681

Kämpfer, P., Busse, H.-J., Horn, H., Abdelmohsen, U. R., Hentschel, U., & Glaeser, S. P. (2016). Williamsia herbipolensis sp. nov., isolated from the phyllosphere of Arabidopsis thaliana. International Journal of Systematic and Evolutionary Microbiology, 66(11), 4609-4613. doi:doi:10.1099/ijsem.0.001398

Klumper, U. et al. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community. ISME J 9, 934-945, doi:10.1038/ismej.2014.191 (2015).

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Knief, C., Frances, L., & Vorholt, J. (2010). Competitiveness of diverse Methylobacterium Strains in the phyllosphere of Arabidopsis thaliana and identification of representative models, including M. extorquens PA1. Microbial Ecology, 60(2), 440-452. doi:10.1007/s00248-010-9725-3

Migula, W. (1895). Über ein neues System der Bakterien.

Miller, E. S., Woese, C. R., & Brenner, S. (1991). Description of the Erythromycin-Producing Bacterium Arthrobacter sp. Strain NRRL B-3381 as Aeromicrobium erythreum gen. nov., sp. nov. International Journal of Systematic and Evolutionary Microbiology, 41(3), 363-368.

Nelson, K., Weinel, C., Paulsen, I., Dodson, R., Hilbert, H., Martins dos Santos, V., . . . Holmes, M. (2002). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environmental Microbiology, 4(12), 799-808.

Orla-Jensen, S. (1919). The Lactic Acid Bacteria. Copenhagen: Fred Host and Son.

Pammel, L. (1895). Bacteriosis of rutabaga (Bacillus campestris n. sp.). Iowa State Coll Agric Exp Stn Bull, 27, 130-134.

PATT, T. E., COLE, G. C., & HANSON, R. S. (1976). Methylobacterium, a New Genus of Facultatively Methylotrophic Bacteria. International Journal of Systematic and Evolutionary Microbiology, 26(2), 226-229. doi:doi:10.1099/00207713-26-2-226

Remus-Emsermann, M. N. P., Kim, E. B., Marco, M. L., Tecon, R., & Leveau, J. H. J. (2013). Draft Genome Sequence of the Phyllosphere Model Bacterium Pantoea agglomerans 299R. Genome Announcements, 1(1). doi:10.1128/genomeA.00036-13

Remus-Emsermann, M. N. P., Schmid, M., Gekenidis, M.-T., Pelludat, C., Frey, J. E., Ahrens, C. H., & Drissner, D. (2016). Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites. Standards in Genomic Sciences, 11(1), 1-12. doi:10.1186/s40793-016-0190-6

Rivas, R., Abril, A., Trujillo, M. E., & Velázquez, E. (2004). Sphingomonas phyllosphaerae sp. nov., from the phyllosphere of Acacia caven in Argentina. International Journal of Systematic and Evolutionary Microbiology, 54(6), 2147-2150.

Steyn, P. L., Segers, P., Vancanneyt, M., Sandra, P., Kersters, K., & Joubert, J. J. (1998). Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. Proposal of the family Sphingobacteriaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 48(1), 165-177. doi:doi:10.1099/00207713-48-1- 165

Takikawa, Y., Serizawa, S., Ichikawa, T., Tsuyumu, S., & Goto, M. (1989). Pseudomonas syringae pv. actinidiae pv. nov

The Causal Bacterium of Canker of Kiwifruit in Japan. Japanese Journal of Phytopathology, 55(4), 437-444. doi:10.3186/jjphytopath.55.437

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Vanneste, J (2000). Fire blight: the disease and its causative agent, Erwinia amylovora. CABI publishing, New York, USA.

Willems, A., De Ley, J., Gillis, M., & Kersters, K. (1991). NOTES: comamonadaceae, a new family encompassing the acidovorans rRNA complex, including variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969). International Journal of Systematic and Evolutionary Microbiology, 41(3), 445-450.

Willems, A., Falsen, E., Pot, B., Jantzen, E., Hoste, B., Vandamme, P., . . . De Ley, J. (1990). Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb. nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov. International Journal of Systematic and Evolutionary Microbiology, 40(4), 384-398.

Yabuuchi, E., Yano, I., Oyaizu, H., Hashimoto, Y., Ezaki, T., & Yamamoto, H. (1990). Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and Two Genospecies of the Genus Sphingomonas. MICROBIOLOGY and IMMUNOLOGY, 34(2), 99-119.

Zgurskaya, H., Evtushenko, L., Akimov, V., & Kalakoutskii, L. (1993). Rathayibacter gen. nov., including the species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus comb. nov., and six strains from annual grasses. International Journal of Systematic and Evolutionary Microbiology, 43(1), 143-149.

Zopf, W. (1891). Ueber Ausscheidung von Fettfarbstoffen (Lipochromen) seitens gewisser Spaltpilze. Berichte der Deutschen Botanischen Gesellschaft, 9(1), 22-28. doi:10.1111/j.1438- 8677.1891.tb05764.x

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