Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10.

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3' end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic.

Development of a Novel Tissue Blot Hybridization Chain Reaction for the Identification of Plant Viruses.

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.

Biology and genetic diversity of phasey bean mild yellows virus, a common virus in legumes in Australia.

This study examined the natural and experimental host range and aphid and graft transmission of the tentative polerovirus phasey bean mild yellows virus (PBMYV). Eleven complete coding sequences from PBMYV isolates were determined from a range of hosts and locations. We found two genetically distinct variants of PBMYV. PBMYV-1 was the originally described variant, and PBMYV-2 had a large putative recombination in open reading frame 5 such that PBMYV-1 and PBMYV-2 shared only 65-66% amino acid sequence identity in the P5 protein. The virus was transmitted by a clonal colony of cowpea aphids (Aphis craccivora) and by grafting with infected scions but was not transmitted by a clonal colony of green peach aphids (Myzus persicae). PBMYV was found in natural infections in 11 host species with a range of symptoms and severity, including seven important grain legume crops from across a wide geographic area in Australia. PBMYV was common and widespread in the tropical weed phasey bean (Macroptilium lathyroides), but it is likely that there are other major alternative hosts for the virus in temperate regions of Australia. The experimental host range of PBMYV included the Fabaceae hosts chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), and phasey bean, but transmissions failed to infect several other members of the families Asteraceae, Cucurbitaceae, Fabaceae and Solanaceae. PBMYV was commonly found in grain legume crops in eastern and western Australia, sometimes at greater than 90% incidence. This new knowledge about PBMYV warrants further assessments of its economic impact on important grain legume crops.

Genetic diversity and recombination between turnip yellows virus strains in Australia.

Disease outbreaks caused by turnip yellows virus (TuYV), a member of the genus Polerovirus, family Luteoviridae, regularly occur in canola and pulse crops throughout Australia. To understand the genetic diversity of TuYV for resistance breeding and management, genome sequences of 28 TuYV isolates from different hosts and locations were determined using high-throughput sequencing (HTS). We aimed to identify the parts of the genome that were most variable and clarify the taxonomy of viruses related to TuYV. Poleroviruses contain seven open reading frames (ORFs): ORF 0-2, 3a, and 3-5. Phylogenetic analysis based on the genome sequences, including isolates of TuYV and brassica yellows virus (BrYV) from the GenBank database, showed that most genetic variation among isolates occurred in ORF 5, followed by ORF 0 and ORF 3a. Phylogenetic analysis of ORF 5 revealed three TuYV groups; P5 group 1 and group 3 shared 45-49% amino acid sequence identity, and group 2 is a recombinant between the other two. Phylogenomic analysis of the concatenated ORFs showed that TuYV is paraphyletic with respect to BrYV, and together these taxa form a well-supported monophyletic group. Our results support the hypothesis that TuYV and BrYV belong to the same species and that the phylogenetic topologies of ORF 0, 3a and 5 are incongruent and may not be informative for species demarcation. A number of beet western yellow virus (BWYV)- and TuYV-associated RNAs (aRNA) were also identified by HTS for the first time in Australia.

Complete Genome Sequence of a New Mastrevirus, Chickpea Redleaf Virus 2, from Australia.

We present here the complete genome sequence of a novel mastrevirus isolated from Cicer arietinum (chickpea) from Australia. We propose the name chickpea redleaf virus 2.

Faba bean polerovirus 1 (FBPV-1); a new polerovirus infecting legume crops in Australia.

A new polerovirus species with the proposed name faba bean polerovirus 1 (FBPV-1) was found in winter legume crops and weeds in New South Wales, Australia. We describe the complete genome sequence of 5,631 nucleotides, containing all putative open reading frames, from two isolates, one from faba bean (Vicia faba) and one from chickpea (Cicer arietinum). FBPV-1 has a genome organization typical of poleroviruses with six open reading frames. However, recombination analysis strongly supports a recombination event in which the 5' portion of FBPV-1, which encodes for proteins P0, P1 and P1-P2, appears to be from a novel parent with a closest nucleotide identity of only 66% to chickpea chlorotic stunt virus. The 3' portion of FBPV-1 encodes for proteins P3, P4 and P3-P5 and shares 94% nucleotide identity to a turnip yellows virus isolate from Western Australia.

LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis.

Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants.

Two Complete Genome Sequences of Phasey Bean Mild Yellows Virus, a Novel Member of the Luteoviridae from Australia.

We present here the complete genome sequences of a novel polerovirus from Trifolium subterraneum (subterranean clover) and Cicer arietinum (chickpea) and compare these to a partial viral genome sequence obtained from Macroptilium lathyroides (phasey bean). We propose the name phasey bean mild yellows virus for this novel polerovirus.

Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus.

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes and pathogenesis-related genes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h postinoculation with S. sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S. sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S. sclerotiorum.

Cadmium stress alters the redox reaction and hormone balance in oilseed rape (Brassica napus L.) leaves.

In order to understand the physiological response of oilseed rape (Brassica napus L.) leaves to cadmium (Cd) stress and exploit the physiological mechanisms involved in Cd tolerance, macro-mineral and chlorophyll concentrations, reactive oxygen species (ROS) accumulation, activities of enzymatic antioxidants, nonenzymatic compounds metabolism, endogenous hormonal changes, and balance in leaves of oilseed rape exposed to 0, 100, or 200 µM CdSO4 were investigated. The results showed that under Cd exposure, Cd concentrations in the leaves continually increased while macro-minerals and chlorophyll concentrations decreased significantly. Meanwhile, with increased Cd stress, superoxide anion (O2(• -)) production rate and hydrogen peroxide (H2O2) concentrations in the leaves increased significantly, which caused malondialdehyde (MDA) accumulation and oxidative stress. For scavenging excess accumulated ROS and alleviating oxidative injury in the leaves, the activity of enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), was increased significantly at certain stress levels. However, with increased Cd stress, the antioxidant enzyme activities all showed a trend towards reduction. The nonenzymatic antioxidative compounds, such as proline and total soluble sugars, accumulated continuously with increased Cd stress to play a long-term role in scavenging ROS. In addition, ABA levels also increased continuously with Cd stress while ZR decreased and the ABA/ZR ratio increased, which might also be providing a protective role against Cd toxicity.

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis.

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 (PsGA2ox2) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1-like homeobox (KNOX) genes, BP, KNAT2 and KNAT6, which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.

Functional analysis of HvSPY, a negative regulator of GA response, in barley aleurone cells and Arabidopsis.

SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA(3)-induced alpha-amylase expression. With the exception of HvSPYDelta1-5, the other deletion proteins were partially active in the barley assay, including HvSPYDelta6-9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity.