No abstract

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool1. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43, which was crossed into bread wheat from the wild grass Thinopyrum elongatum2,3. Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

Nucleotide-binding leucine-rich repeat receptors (NLRs) recognize pathogen effectors to mediate plant disease resistance often involving host cell death. Effectors escape NLR recognition through polymorphisms, allowing the pathogen to proliferate on previously resistant host plants. The powdery mildew effector AVRA13-1 is recognized by the barley NLR MLA13 and activates host cell death. We demonstrate here that a virulent form of AVRA13, called AVRA13-V2, escapes MLA13 recognition by substituting a serine for a leucine residue at the C-terminus. Counterintuitively, this substitution in AVRA13-V2 resulted in an enhanced MLA13 association and prevented the detection of AVRA13-1 by MLA13. Therefore, AVRA13-V2 is a dominant-negative form of AVRA13 and has likely contributed to the breakdown of Mla13 resistance. Despite this dominant-negative activity, AVRA13-V2 failed to suppress host cell death mediated by the MLA13 auto-active MHD variant. Neither AVRA13-1 nor AVRA13-V2 interacted with the MLA13 auto-active variant, implying that the binding moiety in MLA13 that mediates association with AVRA13-1 is altered after receptor activation. We also show that mutations in the MLA13 coiled-coil domain, which were thought to impair Ca 2+ channel activity and NLR function, instead resulted in MLA13 auto-active cell death. The data constitute an important step to define intermediate receptor conformations during NLR activation.

Plant resistance (R) and pathogen avirulence (Avr) gene interactions play key roles in crop resistance to pathogens. Efficient molecular screening tools for crop species are required to define interactions that trigger host resistance and to understand the virulence mechanisms of pathogenic fungi. Avr identification supports genome-based pathogen surveillance to enhance pre-emptive breeding of disease resistant cereal crops. To this end, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes and is applicable to all wheat cultivars tested so far. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to ten Avr candidates. Our assay is based on newly identified Avr/R-induced marker genes which we used to create promoter-luciferase reporter constructs. We combined these reporter constructs with a dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell death. Our new assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that the uptake of our assay by the community will significantly accelerate Avr identification for many wheat pathogens, leading to improved genomic pathogen surveillance and breeding of disease resistant crops.

This level of detail was only accessible because of the high rate of SARS-CoV-2 sequencing, with sequencing attempted on 1438/1793 (80%) of cases and sequences of appropriate quality for downstream analyses recovered from >75% of all cases.• Transmission chains varied in size and duration, with two dominant incursions (ACT.19 and ACT.20) comprising 35% and 53% of all sequenced cases during the study period, respectively.• The ACT.20 outbreak persisted longer, due to specific challenges with implementing public health interventions in the affected populations.• Both major incursions were successfully curbed through stringent public health measures, including the widespread acceptance of COVID-19 vaccines (>95% of the eligible population by the end of the study period).

This a preprint and has not been peer reviewed. Data may be preliminary.

The Australian Capital Territory rapidly responded to an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant on 12 August 2021 with several public health interventions, including a territory-wide lockdown and genomic sequencing. Prior to this date, SARS-CoV-2 had been eliminated locally since July 7, 2020. Sequencing of >75% of cases identified at least 13 independent incursions with onwards spread in the community during the study period, between 12 August and 11 November 2021. Two incursions resulted in the majority of community transmission during this period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. In this study we explore the demographic factors that contributed to the spread of these incursions. The high rates of SARS-CoV-2 sequencing in the Australian Capital Territory and the relatively small population size facilitated detailed investigations of the patterns of virus transmission. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately.