Shane L. Murray

Basal Resistance Against Pseudomonas syringae in Arabidopsis Involves WRKY53 and a Protein with Homology to a Nematode Resistance Protein

Basal resistance is the ultimately unsuccessful plant defense response to infection with a virulent pathogen. It is thought to be triggered by host recognition of pathogen-associated molecular patterns, with subsequent suppression of particular components by pathogen effectors. To identify novel components of Arabidopsis basal resistance against the bacterial pathogen Pseudomonas syringae pv. tomato, microarray expression profiling was carried out on the cirl mutant, which displays enhanced resistance against P. syringae pv. tomato. This identified two genes, At4g23810 and At2g40000, encoding the transcription factor WRKY53 and the nematode resistance protein-like HSPRO2, whose expression was upregulated in cir1 prior to pathogen infection and in wild-type plants after P. syringae pv. tomato infection. WRKY53 and HSPRO2 are positive regulators of basal resistance. Knockout mutants of both genes were more susceptible to P. syringae pv. tomato infection than complemented lines, with increased growth of the pathogen in planta. WRKY53 and HSPRO2 appear to function downstream of salicylic acid and to be negatively regulated by signaling through jasmonic acid and ethylene.

Characterization of a novel, defense-related Arabidopsis mutant, cir1, isolated by luciferase imaging.

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.

MicroRNAs in fruit trees: discovery, diversity and future research directions

Since the first description of microRNAs (miRNAs) 20 years ago, the number of miRNAs identified in different eukaryotic organisms has exploded, largely due to the recent advances in DNA sequencing technologies. Functional studies, mostly from model species, have revealed that miRNAs are major post-transcriptional regulators of gene expression in eukaryotes. In plants, they are implicated in fundamental biological processes, from plant development and morphogenesis, to regulation of plant pathogen and abiotic stress responses. Although a substantial number of miRNAs have been identified in fruit trees to date, their functions remain largely uncharacterised. The present review aims to summarise the progress made in miRNA research in fruit trees, focusing specifically on the economically important species Prunus persica, Malus domestica, Citrus spp, and Vitis vinifera. We also discuss future miRNA research prospects in these plants and highlight potential applications of miRNAs in the on-going improvement of fruit trees.

A constitutive PR‐1::luciferase expression screen identifies Arabidopsis mutants with differential disease resistance to both biotrophic and necrotrophic pathogens

SUMMARY A complex signal transduction network involving salicylic acid, jasmonic acid and ethylene underlies disease resistance in Arabidopsis. To understand this defence signalling network further, we identified mutants that expressed the marker gene PR-1::luciferase in the absence of pathogen infection. These cir mutants all display constitutive expression of a suite of defence-related genes but exhibit different disease resistance profiles to two biotrophic pathogens, Pseudomonas syringae pv. tomato and Peronospora parasitica NOCO2, and the necrotrophic pathogen Botrytis cinerea. We further characterized cir3, which displays enhanced resistance only to the necrotrophic pathogen. Cir3-mediated resistance to B. cinerea is dependent on accumulated salicylic acid and a functional EIN2 protein.

Increased Resistance to Biotrophic Pathogens in the Arabidopsis Constitutive Induced Resistance 1 Mutant Is EDS1 and PAD4-Dependent and Modulated by Environmental Temperature

The Arabidopsis constitutive induced resistance 1 (cir1) mutant displays salicylic acid (SA)-dependent constitutive expression of defence genes and enhanced resistance to biotrophic pathogens. To further characterise the role of CIR1 in plant immunity we conducted epistasis analyses with two key components of the SA-signalling branch of the defence network, ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We demonstrate that the constitutive defence phenotypes of cir1 require both EDS1 and PAD4, indicating that CIR1 lies upstream of the EDS1-PAD4 regulatory node in the immune signalling network. In light of this finding we examined EDS1 expression in cir1 and observed increased protein, but not mRNA levels in this mutant, suggesting that CIR1 might act as a negative regulator of EDS1 via a post-transcriptional mechanism. Finally, as environmental temperature is known to influence the outcome of plant-pathogen interactions, we analysed cir1 plants grown at 18, 22 or 25°C. We found that susceptibility to Pseudomonas syringae pv. tomato (Pst) DC3000 is modulated by temperature in cir1. Greatest resistance to this pathogen (relative to PR-1:LUC control plants) was observed at 18°C, while at 25°C no difference in susceptibility between cir1 and control plants was apparent. The increase in resistance to Pst DC3000 at 18°C correlated with a stunted growth phenotype, suggesting that activation of defence responses may be enhanced at lower temperatures in the cir1 mutant.

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines.

RNA-seq analysis of resistant and susceptible sub-tropical maize lines reveals a role for kauralexins in resistance to grey leaf spot disease, caused by Cercospora zeina

BackgroundCercospora zeina is a foliar pathogen responsible for maize grey leaf spot in southern Africa that negatively impacts maize production. Plants use a variety of chemical and structural mechanisms to defend themselves against invading pathogens such as C. zeina, including the production of secondary metabolites with antimicrobial properties. In maize, a variety of biotic and abiotic stressors induce the accumulation of the terpenoid phytoalexins, zealexins and kauralexins.ResultsC. zeina-susceptible line displayed pervasive rectangular grey leaf spot lesions, running parallel with the leaf veins in contrast to C. zeina-resistant line that had restricted disease symptoms. Analysis of the transcriptome of both lines indicated that genes involved in primary and secondary metabolism were up-regualted, and although different pathways were prioritized in each line, production of terpenoid compounds were common to both. Targeted phytoalexin analysis revealed that C. zeina-inoculated leaves accumulated zealexins and kauralexins. The resistant line shows a propensity toward accumulation of the kauralexin B series metabolites in response to infection, which contrasts with the susceptible line that preferentially accumulates the kauralexin A series. Kauralexin accumulation was correlated to expression of the kauralexin biosynthetic gene, ZmAn2 and a candidate biosynthetic gene, ZmKSL2. We report the expression of a putative copalyl diphosphate synthase gene that is induced by C. zeina in the resistant line exclusively.DiscussionThis study shows that zealexins and kauralexins, and expression of their biosynthetic genes, are induced by C. zeina in both resistant and susceptible germplasm adapted to the southern African climate. The data presented here indicates that different forms of kauralexins accumulate in the resistant and susceptible maize lines in response to C. zeina, with the accumulation of kauralexin B compounds in a resistant maize line and kauralexin A compounds accumulating in the susceptible line.

Kauralexins and zealexins accumulate in sub-tropical maize lines and play a role in seedling resistance to Fusarium verticillioides

Maize is a socially and economically important crop in Africa (and worldwide) that is severely affected by many fungal pathogens. The pathogen Fusarium verticillioides can infect all maize tissue and causes Fusarium ear rot (FER), a disease that greatly reduces quantity and quality of annual maize yields. In response to fungal infection, maize produces kauralexins and zealexins, which are antimicrobial terpenoid phytoalexins that directly reduce the growth of many fungal pathogens including F. verticillioides. This research evaluates the induction of kauralexins and zealexins in F. verticillioides-inoculated sub-tropical maize lines. CML444 (moderately-resistant to FER) and CML144 (susceptible to FER) accumulated both phytoalexin types in seedling roots in response to seed inoculation with F. verticillioides. CML444 control roots also had higher kauralexin levels in comparison to CML144, suggesting that kauralexin accumulation may be primed for rapid up-regulation in CML444 following fungal infection. The an2 maize mutant, which displays reduced expression of the key kauralexin biosynthetic gene ZmAn2, accumulated decreased root levels of kauralexins and zealexins following F. verticillioides inoculation in comparison to wildtype line W22, confirming that both kauralexins and zealexins play a role in the resistance response to seedling disease caused by F. verticillioides.

Trichoderma asperellum isolated from African maize seed directly inhibits Fusarium verticillioides growth in vitro

Maize is a globally important crop that is affected by fungal diseases causing yield losses annually. One fungus, Fusarium verticillioides, causes the disease Fusarium Ear Rot (FER), which reduces grain quality and produces mycotoxins called fumonisins that are harmful to animals and humans. As chemical fungicides are expensive and have negative environmental effects, the use of biological control agents (BCAs) has become favourable in recent years. A commonly used fungal BCA is Trichoderma spp., which has been effective in reducing disease incidence as well as enhancing crop growth. In this study, T. asperellum was isolated from an African maize line and was shown to significantly inhibit growth of F. verticillioides in an in vitro competition assay.