Alison C. Jackson

A local regulatory network around three NAC transcription factors in stress responses and senescence in Arabidopsis leaves

Summary A model is presented describing the gene regulatory network surrounding three similar NAC transcription factors that have roles in Arabidopsis leaf senescence and stress responses. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1-hybrid analysis and modelling using gene expression time course data has been applied to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modelling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions. Gene expression analysis in mutants of ANAC019 and ANAC055 at different times during leaf senescence has revealed a distinctly different role for each of these genes. Yeast 1-hybrid analysis is shown to be a valuable tool that can distinguish clades of binding proteins and be used to test and quantify protein binding to predicted promoter motifs.

Can ABA mediate responses of salinity stressed tomato

We tested the hypothesis that responses to root-sourced salt-stress, in the root and shoot, are coordinated by elevated abscisic acid (ABA) concentrations. To do this we examined the responses of wild-type (Ailsa Craig) and ABA-deficient mutant (notabilis) genotypes of tomato to a range of salinity (0 (control), 30, 60, 90, 120 mM NaCl). Leaf area was reduced and root:shoot ratio increased in response to increasing salinity in the absence of changes in leaf turgor for both genotypes. Stomatal conductance was however reduced suggesting that a non-hydraulic factor may be coordinating responses to the salinity in the rooting medium. ABA concentrations were increased in response to salinity and correlative analysis showed that stomatal conductance exhibited a negative relationship with increasing xylem ABA for both genotypes (P<0.001). The possibility that ABA controlled leaf area was less apparent as reduced leaf areas were found in both wild-type and notabilis under high salinity (120 mM; P<0.001), but xylem sap and tissue ABA concentrations were much lower in notabilis. More specific evidence for the role of enhanced ABA concentrations on leaf and root growth was found under moderate levels of salinity. At 90 mM NaCl there was a large decrease in leaf area for notabilis compared with the near control levels of leaf area exhibited by the wild-type (P<0.001). We hypothesised that the poorer growth in the ABA-deficient mutant was due to its inability to produce ABA at wild-type levels. This was confirmed, by feeding synthetic ABA to the rooting medium, and that 1 and 10 μM±ABA appeared necessary for the maintenance of leaf and root growth at wild-type levels, respectively. These data suggested that there was differential sensitivity to ABA in the roots and shoots but that enhanced concentrations at wild-type levels acted primarily to maintain root and shoot growth under moderate salt stress. How ABA was synthesised to affect concentrations within the leaf and root tissue under salinity stress was investigated by the analysis of mRNAs for two enzymes involved in ABA biosynthesis (9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP). Evidence for increased synthesis in the roots was detected but there was little change in the leaves in response to salinity and alternative mechanisms that may regulate ABA content in the leaf are explored. The possibility that ABA can interact with other hormones to coordinate whole plant responses to high salinity stress is discussed.

TEMPRANILLO is a regulator of juvenility in plants

Many plants are incapable of flowering in inductive daylengths during the early juvenile vegetative phase (JVP). Arabidopsis mutants with reduced expression of TEMPRANILLO (TEM), a repressor of FLOWERING LOCUS T (FT) had a shorter JVP than wild-type plants. Reciprocal changes in mRNA expression of TEM and FT were observed in both Arabidopsis and antirrhinum, which correlated with the length of the JVP. FT expression was induced just prior to the end of the JVP and levels of TEM1 mRNA declined rapidly at the time when FT mRNA levels were shown to increase. TEM orthologs were isolated from antirrhinum (AmTEM) and olive (OeTEM) and were expressed most highly during their juvenile phase. AmTEM functionally complemented AtTEM1 in the tem1 mutant and over-expression of AmTEM prolonged the JVP through repression of FT and CONSTANS (CO). We propose that TEM may have a general role in regulating JVP in herbaceous and woody species.

Identification of pathogenicity-related genes in Fusarium oxysporum f. sp. cepae

Summary Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.

Characterization of isolates that cause black rot of crucifers in East Africa

A study was conducted in the East African countries of Kenya, Tanzania and Uganda in the months of July and August 2009 with the objectives of assessing the status of black rot and race structure of Xanthomonas campestris pv. campestris in the three countries. Samples infected with black rot were collected from farmers’ fields mainly from Brassica oleracea crops (broccoli, cabbage, cauliflower and kales). A total of 399 farms were surveyed of which 260 were from Kenya, 91 from Tanzania and 48 from Uganda. Following successful isolations, a total of 249 isolates of the causal agent, Xanthomonas campestris pv. campestris were recovered. Pathogenicity of all isolates was confirmed on B. oleracea susceptible cultivars Copenhagen Market F1 and Wirosa F1. Sixty of the 250 isolates were race-typed using a differential set Brassica spp. Only two races, 1 (Kenya and Tanzania) and 4 (Kenya, Tanzania and Uganda) were observed however, another race (5) was observed from one isolate recovered from a B. rapa sample obtained from Tanzania in 2003. Genomic fingerprinting with repetitive-PCR revealed clusters that did not depict significant correlations between isolates and geographical location, isolates and host adaptation or isolates and race. However, it did demonstrate existence of genetic differences within the East African X. campestris pv. campestris population indicating that it is not a similar clonal population of the same genetic background.

Pangenomic analysis reveals pathogen-specific regions and novel effector candidates in Fusarium oxysporum f.sp. cepae

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

First Report of Fusarium oxysporum Causing a Vascular Wilt of Statice (Limonium sinuatum) in the U.K.

Statice (Limonium sinuatum) is grown commercially in many countries as a cut-flower crop. Fungal and oomycete pathogens reported for this plant include Colletotrichum, Botrytis, Cercospora, Rhizoctonia, and Peronospora (Moorman 2016). In the UK, 80% of all statice production (2.5 ha, value

Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid.

800,000) is through a specialist grower in Lincolnshire. In 2016, 30% of the 70,000 statice plants raised at this nursery were affected by a vascular wilt. Symptoms observed on approximately 1 m tall flowering plants (cv. Velvet Wings) consisted of wilted leaves and flower stalks, often progressing down one side of the stem, followed by leaf necrosis and plant death. Brown staining of vascular tissue was observed in dissected stems and, in advanced infections, mycelium was evident at the stem base. Isolations were carried out from 18 plants by excising 2 to 3 cm sections of infected stem, surface disinfecting in 5% sodium hypochlorite for 2 min, rinsing in sterile water and plating on potato dextrose agar (PDA) containing 20 µg/ml chlortetracycline. After incubation at 20°C for 4 days under natural light, fungal colonies were sub-cultured onto PDA and grown for a further 10 days. Cultures displayed morphology typical of Fusarium species, with a peach/pale pink color. Macroconidia were 3-septate, straight or slightly curved, 16.1 to 25.7 µm x 2.8 to 5.4 µm. Microconidia were abundant, elliptical or reniform, 6.1 to 12.4 µm x 1.6 to 4.9 µm. DNA was extracted from four isolates, and the translation elongation factor 1-α (EF1-α) gene amplified by PCR and sequenced using exTEF-F/ FUexTEF-R primers (Taylor et al. 2016). All isolates shared identical EF1-α sequences (GenBank accession number KX822794) and were confirmed as Fusarium oxysporum using BLAST analysis. The sequences were 99% identical to the EF1- α sequences of a wide range of F. oxysporum isolates with the greatest homology to F. oxysporum f. sp. freesia (KP964900, 99.7% over 989 bp). To confirm pathogenicity, statice (cv. Velvet Wings) plants produced by tissue culture were raised in pots. Once the first flower stalk reached approximately 30 cm, plants were removed, the bottom third of the root system excised and the remaining roots soaked in a conidial suspension (1 x 106 conidia/ml) for 5 min. As a negative control treatment, F. oxysporum isolate Fo47, a biocontrol strain known to be non-pathogenic on a range of hosts (Aime et al. 2013), was used to inoculate plants in the same way, while noninoculated control plants were soaked in sterile water. All statice (six plants / isolate or control treatment) were then replanted in compost and pots placed in a randomized design in a temperature controlled glasshouse (25°C day, 18°C night, 16 hour photoperiod). After 20 days, typical wilt symptoms were observed in all the plants inoculated with the four F. oxysporum isolates from statice, and after 40 days all the plants were dead (e-Xtra). All noninoculated control plants and those inoculated with F. oxysporum isolate Fo47 remained healthy. The pathogen was successfully re-isolated from infected stems with the fungal cultures exhibiting the same morphology as described previously. Furthermore, following DNA extraction, amplification and sequencing of the EF1-α gene of the re-isolated fungi, all sequences were 100% identical to those obtained for the original isolates, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of Fusarium wilt of statice. Further work is required to establish the host range of this potentially new forma specialis.