Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.

The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.

Multiplex CRISPR-Cas9 Gene-Editing Can Deliver Potato Cultivars with Reduced Browning and Acrylamide.

Storing potato tubers at cold temperatures, either for transport or continuity of supply, is associated with the conversion of sucrose to reducing sugars. When cold-stored cut tubers are processed at high temperatures, with endogenous asparagine, acrylamide is formed. Acrylamide is classified as a carcinogen. Potato processors prefer cultivars which accumulate fewer reducing sugars and thus less acrylamide on processing, and suitable processing cultivars may not be available. We used CRISPR-Cas9 to disrupt the genes encoding vacuolar invertase (VInv) and asparagine synthetase 1 (AS1) of cultivars Atlantic and Desiree to reduce the accumulation of reducing sugars and the production of asparagine after cold storage. Three of the four guide RNAs employed induced mutation frequencies of 17-98%, which resulted in deletions, insertions and substitutions at the targeted gene sites. Eight of ten edited events had mutations in at least one allele of both genes; for two, only the VInv was edited. No wild-type allele was detected in both genes of events DSpco7, DSpFN4 and DSpco12, suggesting full allelic mutations. Tubers of two Atlantic and two Desiree events had reduced fructose and glucose concentrations after cold storage. Crisps from these and four other Desiree events were lighter in colour and included those with 85% less acrylamide. These results demonstrate that multiplex CRISPR-Cas9 technology can generate improved potato cultivars for healthier processed potato products.

Enabling Trade in Gene-Edited Produce in Asia and Australasia: The Developing Regulatory Landscape and Future Perspectives.

Genome- or gene-editing (abbreviated here as 'GEd') presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world's growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Small RNA Analyses of a Ceratobasidium Isolate Infected with Three Endornaviruses.

Isolates of three endornavirus species were identified co-infecting an unidentified species of Ceratobasidium, itself identified as a symbiont from within the roots of a wild plant of the terrestrial orchid Pterostylis vittata in Western Australia. Isogenic lines of the fungal isolate lacking all three mycoviruses were derived from the virus-infected isolate. To observe how presence of endornaviruses influenced gene expression in the fungal host, we sequenced fungus-derived small RNA species from the virus-infected and virus-free isogenic lines and compared them. The presence of mycoviruses influenced expression of small RNAs. Of the 3272 fungus-derived small RNA species identified, the expression of 9.1% (300 of 3272) of them were up-regulated, and 0.6% (18 of 3272) were down-regulated in the presence of the viruses. Fourteen novel micro-RNA-like RNAs (Cer-milRNAs) were predicted. Gene target prediction of the differentially expressed Cer-milRNAs was quite ambiguous; however, fungal genes involved in transcriptional regulation, catalysis, molecular binding, and metabolic activities such as gene expression, DNA metabolic processes and regulation activities were differentially expressed in the presence of the mycoviruses.

Fungal endophytes from salt-adapted plants confer salt tolerance and promote growth in wheat (Triticum aestivum L.) at early seedling stage.

With increasing human global population, increased yield under saline conditions is a desirable trait for major food crops. Use of endophytes, isolated from halophytic hosts, seems to be an exciting approach for conferring salt tolerance to a salt-sensitive crop. Therefore, in the current study, fungal endophytes were isolated from halophytic plants' roots and their ability to withstand in vitro salt stress was evaluated. The fungal endophytes could withstand up to 1M NaCl concentrations and this tolerance was independent of their host or tissue source. When inoculated on salt-sensitive wheat seeds/seedlings, several of the endophytes showed a positive impact on germination and biomass-related parameters upon salt stress, both in vitro and under glasshouse conditions. One of the isolates from dicot plants (identified as Microsphaeropsis arundinis) could successfully colonize wheat and promote its growth under salt and no-salt conditions. Amongst the fungal isolates that are known to be natural endophytes of wheat, Chaetomium globosum was the best performing isolate and has previously been reported to be an effective biocontrol agent. Based on the results of our preliminary study, we suggest that these fungal endophytes could prove beneficial for enhancing the salt stress tolerance of wheat crop.

Spillover of a Tobamovirus from the Australian Indigenous Flora to Invasive Weeds.

The tobamovirus yellow tailflower mild mottle virus (YTMMV) was previously reported in wild plants of Anthocercis species (family Solanaceae) and other solanaceous indigenous species growing in natural habitats in Western Australia. Here, we undertook a survey of two introduced solanaceous weeds, namely Solanum nigrum (black nightshade) and Physalis peruviana (cape gooseberry) in the Perth metropolitan area and surrounds to determine if YTMMV has spread naturally to these species. At a remnant natural bushland site where both solanaceous weeds and indigenous Anthocercis hosts grew adjacent to one another, a proportion of S. nigrum and P. peruviana plants were asymptomatically-infected with YTMMV, confirming spillover had occurred. Populations of S. nigrum also grow as weeds in parts of the city isolated from remnant bushland and indigenous sources of YTMMV, and some of these populations were also infected with YTMMV. Fruit was harvested from virus-infected wild S. nigrum plants and the seed germinated under controlled conditions. Up to 80% of resultant seedlings derived from infected parent plants were infected with YTMMV, confirming that the virus is vertically-transmitted in S. nigrum, and therefore infection appears to be self-sustaining in this species. This is the first report of spillover of YTMMV to exotic weeds, and of vertical transmission of this tobamovirus. We discuss the roles of vertical and horizontal transmission in this spillover event, and its implications for biosecurity.

Differential Symptom Development and Viral RNA Loads in 10 Nicotiana benthamiana Accessions Infected with the Tobamovirus Yellow Tailflower Mild Mottle Virus.

Yellow tailflower mild mottle virus (YTMMV, genus Tobamovirus) was identified from wild plants of solanaceous species in Australia. Nicotiana benthamiana is a species indigenous to the arid north of Australia. N. benthamiana accession RA-4 (the lab type), which has a mutant, functionally defective, RNA-dependent RNA polymerase 1 (Rdr1) gene (Nb-Rdr1m), has played a significant role in plant virology, but little study has been done regarding responses to virus infection by other accessions of N. benthamiana. All wild-collected N. benthamiana accessions used in this study harbored wild-type Rdr1 genes (Nb-Rdr1). We compared symptoms of YTMMV infection and viral RNA load on RA-4 and nine wild-collected accessions of N. benthamiana from mainland Western Australia, an island, and the Northern Territory. After inoculation with YTMMV, RA-4 plants responded with systemic hypersensitivity and all individuals were dead 35 days postinoculation (dpi). Plants of wild-collected accessions exhibited a range of symptoms, from mild to severe, and some, but not all, died in the same period. Quantitative reverse transcription PCR revealed that the Rdr1 mutation was not a predictor of viral RNA load or symptom severity. For example, wild-collected A019412 plants carried more than twice the viral RNA load of RA-4 plants, but symptom expression was moderate. For plants of most accessions, viral RNA load did not increase after 10 dpi. The exception was plants of accession Barrow-1, in which viral RNA load was low until 15 dpi, after which it increased more than 29-fold. This study revealed differential responses by N. benthamiana accessions to infection by an isolate of YTMMV. The Rdr1 gene, whether mutant or wild-type, did not appear to influence viral RNA load or disease expression. Genetic diversity of the 10 N. benthamiana accessions in some cases reflected geographical location, but in other accessions this was not so.

Genome-Wide Identification and Validation of Target Genes Associated with Insecticide Treatment of the Green Peach Aphid, Myzus persicae.

Next-generation sequencing and analyses of whole-genome transcripts can be used to identify genes and potential mechanisms that may be responsible for the development of resistance to insecticides. Such genes can be identified by isolating and sequencing high-quality messenger RNA and identifying differentially expressed genes (DEGs), and gene variants from insecticide-treated and untreated colonies of the Green peach aphid (GPA) or resistant and susceptible GPA populations. Datasets generated would reveal a set of genes whose expression may be associated with the insecticide treatment. The DEGs can then be validated using quantitative PCR assays.

Functional Characterization of Target Genes Associated with Insecticide Resistance of the Green Peach Aphid, Myzus persicae.

Identifying genes responsive to insecticide treatment is the first step towards understanding the mechanism(s) of insecticide resistance and the development of effective insecticides against economic insect pests such as the Green peach aphid (GPA). Functional and Reverse Genetics approaches such as the RNA interference (RNAi) technology can be used to assess the possible involvement of genes whose expression is associated with an insecticide treatment. For GPA, this can be done by comparing the behavior and development of the insect following RNAi of a putative gene associated with insecticide treatment and exposure of the RNAi-treated insects to lethal doses of insecticides. In a case where knockdown of a gene or genes increases the susceptibility of RNAi-treated populations compared to controls, the target gene may have a direct role in the development of resistance to the insecticide or the gene may be involved in other metabolic processes that may be required for resilience against the insecticide.

RNA interference of an orthologue of Dicer of Meloidogyne incognita alludes to the gene's importance in nematode development.

Dicers and dicer-like enzymes play an essential role in small RNA processing in eukaryotes. Nematodes are thought to encode one dicer, DCR-1; only that for Caenorhabditis spp. is well-characterised. Using genomic sequences of eight root-knot nematodes (Meloidogyne spp.), we identified putative coding sequences typical of eukaryotic DICERS. We noted that the primary and secondary structures of DICERS they encode were different for different Meloidogyne species and even for isolates of the same species, suggesting paralogy for the gene. One of the genes for M. incognita (Midcr-1.1) expressed in eggs, juvenile stage 2 and adults, with the highest expression in the adult females. All the Meloidogyne DICERS had seven major domains typical of those for Caenorhabditis spp. and humans with very similar protein folding. RNAi of Midcr-1.1 in J2s using seven dsRNAs, each based on sequences encoding the domains, induced mild paralysis but measurable knockdown was detected in J2s treated with five of the dsRNAs. For four of the dsRNAs, the RNAi effect lasted and reduced the nematode's infectivity. Also, host plant delivery of dsRNAs complementary to coding sequences of the Dicer Dimerisation domain impaired development, reducing nematode infection by 71%. These results confirm the importance of the gene to nematode health.

Attempt to Silence Genes of the RNAi Pathways of the Root-Knot Nematode, Meloidogyne incognita Results in Diverse Responses Including Increase and No Change in Expression of Some Genes.

Control of plant-parasitic nematodes (PPNs) via host-induced gene silencing (HIGS) involves rational selection of genes and detailed assessment of effects of a possible knockdown on the nematode. Some genes by nature may be very important for the survival of the nematode that knockdown may be resisted. Possible silencing and effects of 20 such genes involved in the RNA interference (RNAi) pathways of Meloidogyne incognita were investigated in this study using long double-stranded RNAs (dsRNAs) as triggers. Two of the genes, ego-1 and mes-2, could not be knocked down. Expression of six genes (xpo-1, pash-1, xpo-2, rha-1, ekl-4, and csr-1) were significantly upregulated after RNAi treatment whereas for 12 of the genes, significant knockdown was achieved and with the exception of mes-2 and mes-6, RNAi was accompanied by defective phenotypes in treated nematodes including various degrees of paralysis and abnormal behaviors and movement such as curling, extreme wavy movements, and twitching. These abnormalities resulted in up to 75% reduction in infectivity of a tomato host, the most affected being the J2s previously treated with dsRNA of the gfl-1 gene. For 10 of the genes, effects of silencing in the J2s persisted as the adult females isolated from galls were under-developed, elongated, and transparent compared to the normal saccate, white adult females. Following RNAi of ego-1, smg-2, smg-6, and eri-1, reduced expression and/or the immediate visible effects on the J2s were not permanent as the nematodes infected and developed normally in tomato hosts. Equally intriguing was the results of RNAi of the mes-2 gene where the insignificant change in gene expression and behavior of treated J2s did not mean the nematodes were not affected as they were less effective in infecting host plants. Attempt to silence drsh-1, mut-7, drh-3, rha-1, pash-1, and vig-1 through HIGS led to reduction in nematode infestation by up to 89%. Our results show that genes may respond to RNAi knockdown differently so an exhaustive assessment of target genes as targets for nematode control via RNAi is imperative.

Challenges to elucidating how endornaviruses influence fungal hosts: Creating mycovirus-free isogenic fungal lines and testing them.

Determining roles of mycoviruses in fungal biology is complicated, especially when fungi are co-infected with multiple viruses. Genetically identical (isogenic) fungal lines that are infected by and not infected by viruses must be created and compared. Here, we study an isolate of Ceratobasidium sp., a fungus isolated from pelotons in roots of a wild terrestrial orchid. The fungal isolate was co-infected with three distinct endornaviruses, isolates of Ceratobasidium endonarvirus B (CbEVB), Ceratobasidium endonarvirus C (CbEVC) and Ceratobasidium endonarvirus D (CbEVD). An experiment to reveal natural distribution of the three mycoviruses within a fungal colony revealed no sectoring; they were all evenly distributed throughout the colony. Hyphal tipping and treatments with one of five antibiotics (kanamycin, streptomycin, cycloheximide, rifampicin and ampicillin) were applied in attempts to 'cure' fungal lines of one, two or three of the viruses present. Surprisingly, the three mycoviruses responded differentially to each curing approach. The isolate of CbEVC was eliminated upon treatment with cycloheximide, but not with kanamycin or streptomycin, whereas the isolate of CbEVD did not respond to cycloheximide. The isolate of CbEVB was eliminated upon all treatments. In some cases, a virus was undetectable by species-specific RT-PCR assay after treatment, but when the fungus was cultured for a period on non-selective medium, the virus was detected again. Effects of mycoviruses on growth characteristics of isogenic fungal lines on two nutrient media were studied. Co-infection by the three viruses reduced mycelial growth rate on both media. In contrast, some fungal lines infected with one or two mycoviruses grew more rapidly than virus-free lines.

Co-Infection with Three Mycoviruses Stimulates Growth of a Monilinia fructicola Isolate on Nutrient Medium, but Does Not Induce Hypervirulence in a Natural Host.

Monilinia fructicola and Monilinia laxa are the most destructive fungal species infecting stone fruit (Prunus species). High-throughput cDNA sequencing of M. laxa and M. fructicola isolates collected from stone fruit orchards revealed that 14% of isolates were infected with one or more of three mycoviruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). Isolate M196 of M. fructicola was co-infected with all three viruses, and this isolate was studied further. Several methods were applied to cure M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. When isogenic fungal lines were cultured on nutrient agar medium in vitro, the triple mycovirus-infected parent isolate M196 grew 10% faster than any of the virus-cured isogenic lines. BVF had a slight inhibitory effect on growth, and FPV1 did not influence growth. Surprisingly, after inoculation to fruits of sweet cherry, there were no significance differences in disease progression between isogenic lines, suggesting that these mycoviruses did not influence the virulence of M. fructicola on a natural host.

Serendipitous identification of Pratylenchus curvicauda from the grainbelt of Western Australia.

A Pratylenchus species identified during a survey of Pratylenchus quasitereoides incidence at four locations of the grainbelt of Western Australia is described. Morphological and morphometric features indicated the previously undescribed morphotypes in nematode mixtures encountered were conspecific to P. curvicauda, and were clearly distinguishable from nine common Pratylenchus spp. Typical features of P. curvicauda were its body length (415-540 µm), which was curved to a c-shaped with a maximum body diameter of 20 µm, and the nature of its tail; 34 µm long, 2.8 µm wide at the anus and a typical ventrally arcuate with a round terminus. Sequenced for the first time, the sequences of the partial 18S-ITS1-5.8S-ITS2-partial 28S (80 clones, 14 individual nematodes) and the 28S-D3 (17 clones) regions of the rDNA of P. curvicauda had overall mean distances of 0.013 and 0.085, respectively. Phylogenetic analyses with sequences of both segments of the rDNA clearly showed the P. curvicauda isolates as monophyletic, distinct from ca 40 Pratylenchus species. Notably, it was distinct from Pratylenchus species present in Australia including P. quasitereoides and a Western Australia isolate of P. thornei. Further research into the biology of P. curvicauda is needed to facilitate development of strategies for its management, if it is an important pest.

Host Specificity of Endophytic Mycobiota of Wild Nicotiana Plants from Arid Regions of Northern Australia.

In arid regions of northern Australia, plants survive under water deficit, high temperatures, intense solar radiation and nutrient-impoverished soils. They employ various morpho-physiological and biochemical adaptations including interaction with microbial symbionts. We evaluated identity, host and tissue association with geographical distribution of fungal endophytes isolated from above- and below-ground tissues of plants of three indigenous Australian Nicotiana species. Isolation frequency and α-diversity were significantly higher for root endophyte assemblages than those of stem and leaf tissues. We recorded no differences in endophyte species richness or diversity as a function of sampling location, but did detect differences among different host genotypes and plant tissues. There was a significant pattern of community similarity associated with host genotypes but no consistent pattern of fungal community structuring associated with sampling location and tissue type, regardless of the community similarity measurements used.

Novel and divergent viruses associated with Australian orchid-fungus symbioses.

Terrestrial orchids represent a symbiotic union between plants and mycorrhizal fungi. This study describes the occurrence and nature of viruses associated with one population of wild Pterostylis sanguinea orchids, including their fungal symbionts, over two consecutive years. A generic sequencing approach, which combined dsRNA-enrichment from plant and mycelial tissues, random amplification and high throughput shotgun sequencing was used to identify novel viruses. The majority of the virus-like sequences represent partial genomes, and their identification is based solely on de novo assembly of sequencing data. In orchid leaf tissues we found three isolates of a novel totivirus and an unclassified virus; both resemble fungus-infecting viruses. Two isolates of Ceratobasidium sp that were isolated from orchid underground stems contained at least 20 viruses, 16 of which were previously described as alphapartitiviruses and betapartitiviruses. A novel hypovirus and a mitovirus were genetically distant from existing members of the genera and did not readily fit into recognised subgroups.

The challenges of using high-throughput sequencing to track multiple bipartite mycoviruses of wild orchid-fungus partnerships over consecutive years.

The bipartite alpha- and betapartitiviruses are recorded from a wide range of fungi and plants. Using a combination of dsRNA-enrichment, high-throughput shotgun sequencing and informatics, we report the occurrence of multiple new partitiviruses associated with mycorrhizal Ceratobasidium fungi, themselves symbiotically associated with a small wild population of Pterostylis sanguinea orchids in Australia, over two consecutive years. Twenty-one partial or near-complete sequences representing 16 definitive alpha- and betapartitivirus species, and further possible species, were detected from two fungal isolates. The majority of partitiviruses occurred in fungal isolates from both years. Two of the partitiviruses represent phylogenetically divergent forms of Alphapartitivirus, suggesting that they may have evolved under long geographical isolation there. We address the challenge of pairing the two genomic segments of partitiviruses to identify species when multiple partitiviruses co-infect a single host.

A simple and rapid in vitro test for large-scale screening of fungal endophytes from drought-adapted Australian wild plants for conferring water deprivation tolerance and growth promotion in Nicotiana benthamiana seedlings.

Some fungal endophytes confer novel phenotypes and enhance existing ones in plants, including tolerance to water deprivation stress. A range of fungal endophytes was isolated from wild Nicotiana plants growing in arid parts of northern Australia. These were screened for ability to enhance water deprivation stress tolerance by inoculating seedlings of the model plant N. benthamiana in two in vitro tests. Sixty-eight endophyte isolates were co-cultivated with N. benthamiana seedlings on either damp filter paper or on agar medium before being subjected to water deprivation. Seventeen isolates were selected for further testing under water deprivation conditions in a sand-based test in a glasshouse. Only two fungal isolates, Cladosporium cladosporioides (E-162) and an unknown fungus (E-284), significantly enhanced seedling tolerance to moisture deprivation consistently in both in vitro and sand-based tests. Although a strongly significant correlation was observed between any two screening methods, the result of filter paper test was more strongly reflected (r = 0.757, p < 0.001) in results of the glasshouse test, indicating its relative suitability over the agar-based test. In another experiment, the same 17 isolates carried forward to the sand-based test used in the glasshouse screening test were inoculated to N. benthamiana plants in pots in a nutrient-limiting environment to test their influence on growth promotion. Isolates related to C. cladosporioides, Fusarium equiseti, and Thozetella sp. promoted seedling growth by increasing shoot length and biomass. The fungal isolate E-162 (C. cladosporioides) significantly enhanced moisture deprivation tolerance as well as promoted seedling growth.

Evolution of a wild-plant tobamovirus passaged through an exotic host: Fixation of mutations and increased replication.

Tobamovirus is a group of viruses that have become serious pathogens of crop plants. As part of a study informing risk of wild plant virus spill over to crops, we investigated the capacity of a solanaceous-infecting tobamovirus from an isolated indigenous flora to adapt to new exotic hosts. Yellow tailflower mild mottle virus (YTMMV) (genus Tobamovirus, family Virgaviridae) was isolated from a wild plant of yellow tailflower (Anthocercis littoria, family Solanaceae) and initially passaged through a plant of Nicotiana benthamiana, then one of Nicotiana glutinosa where a single local lesion was used to inoculate a N. benthamiana plant. Sap from this plant was used as starting material for nine serial passages through three plant species. The virus titre was recorded periodically, and 85% of the virus genome was sequenced at each passage for each host. Six polymorphic sites were found in the YTMMV genome across all hosts and passages. At five of these, the alternate alleles became fixed in the viral genome until the end of the experiment. Of these five alleles, one was a non-synonymous mutation (U1499C) that occurred only when the virus replicated in tomato. The mutant isolate harbouring U1499C, designated YTMMV-δ, increased its titre over passages in tomato and outcompeted the wild-type isolate when both were co-inoculated to tomato. That YTMMV-δ had greater reproductive fitness in an exotic host than did the wild type isolate suggests YTMMV evolution is influenced by host changes.