Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly evolved from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This evolution has been facilitated by the dramatic drop in costs, advances in technology, and concerted efforts to translate sequencing data into actionable information. There is however a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonisation, comparability and validation. In this review, we outline the current landscape of WGS pipelines and applications and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repository of resistance-causing variants, phylogenetic analyses, quality control processes, and standardised reporting. 1. Introduction Mycobacterium tuberculosis complex (Mtbc) pathogens are collectively the top infectious disease killer globally, causing 10 million new tuberculosis (TB) cases annually 1. Increasingly, 95 new TB cases are already resistant to rifampicin and isoniazid (termed multidrug resistance; 96 MDR-TB), the key first line drugs 1. Tackling the spread and drug resistance burden of this pathogen requires concerted global effort in prevention, diagnosis, treatment and surveillance.

In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3 a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 alleledependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogenencoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes.

Summary There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race‐specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3 a2/f2 that is recognized by Pm3a/f was known molecularly.We performed map‐based cloning and genome‐wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2.The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase‐like effectors, including Avr a13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13.These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

The wheat Pm3 resistance gene against the powdery mildew pathogen occurs as an allelic series encoding functionally different immune receptors which induce resistance upon recognition of isolate-specific avirulence (AVR) effectors from the pathogen. Here, we describe the identification of five effector proteins from the mildew pathogens of wheat, rye, and the wild grass Dactylis glomerata , specifically recognized by the PM3B, PM3C and PM3D receptors. Together with the earlier identified AVRPM3 A2/F2 , the recognized AVRs of PM3B/C, (AVRPM3 B2/C2 ), and PM3D (AVRPM3 D3 ) belong to a large group of proteins with low sequence homology but predicted structural similarities. AvrPm3 b2/c2 and AvrPm3 d3 are conserved in all tested isolates of wheat and rye mildew, and non-host infection assays demonstrate that Pm3b , Pm3c , and Pm3d are also restricting the growth of rye mildew on wheat. Furthermore, divergent AVR homologues from non-adapted rye and Dactylis mildews are recognized by PM3B, PM3C, or PM3D, demonstrating their involvement in host specificity.