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Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function.

Blackleg disease, caused by the ascomycete fungal pathogen Leptosphaeria maculans, is a devastating disease of canola (Brassica napus) in Australia, Canada and Europe. The pathogen is considered a global invasive species and poses a threat to canola production in China, where only the weakly aggressive strain L. biglobosa is present. In Canada, breakdown of blackleg resistance has been shown. In order to develop a more effective disease management strategy, there is a need to elucidate host resistance and defense mechanisms underlying the B. napus-L. maculans pathosystem. This is the very first study to investigate major resistance genes (R genes) and adult plant resistance (APR) in Canadian canola germplasm. This study also analyzed the avirulence allele frequency in L. maculans populations in western Canada. R genes were detected in the majority of these B. napus germplasm, with the Rlm3 gene being predominant. The frequency of AvrLm3 allele in field fungal populations was extremely low. APR was identified in more than 50% of the germplasm. This indicated the breakdown of Rlm3 resistance, which could be due to the widespread use of this single resistance gene in Canadian B. napus germplasm and varieties. To address concerns of introducing L. maculans from Canada into China, this study further characterized R genes and APR to L. maculans in a collection of Chinese B. napus germplasm. R genes were detected in more than 40% of the germplasm tested, with Rlm3 and Rlm4 being predominant. A large portion of the Chinese germplasm showed high to moderate ii APR in field trials at three locations in MB, SK and AB in western Canada. This study highlighted the availability of fair to good resistance in the Chinese B. napus germplasm against blackleg disease and was the first study to investigate a large number of Chinese germplasm against Canadian fungal populations in different environments. RNA sequencing of resistant and susceptible host tissues and a streamlined bioinformatics pipeline identified unique genes and plant defense pathways specific to plant resistance in the B. napus-L. maculans LepR1-AvrLepR1 interaction. The sequencing data coupled with functional characterization of some unique genes, in depth histological analysis, and in situ gene activity analysis directly at the site of infection provide unprecedented spatial and temporal resolution of the plant defense response to L. maculans.

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence-absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B. napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates.

The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA- and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA- and PMA-based methods in detecting live bacterial cells in water.

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race-specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co-segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re-sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5-9 to reflect the dual specificity of this locus. Avrlm5-9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5-9 during infection was monitored by RNA sequencing. The recognition of AvrLm5-9 by Rlm9 is masked in the presence of AvrLm4-7, another Lm effector. AvrLm5-9 and AvrLm4-7 do not interact, and AvrLm5-9 is expressed in the presence of AvrLm4-7. AvrLm5-9 is the second Lm effector for which host recognition is masked by AvrLm4-7. An understanding of this complex interaction will provide new opportunities for the engineering of broad-spectrum recognition.

Leptosphaeria maculans is the causal agent of blackleg, a serious disease on canola/rapeseed in western Canada, Australia and Europe. Genetic resistance and extended crop rotation provided effective disease control in western Canada for years but the emergence of new pathogen races has reduced the effectiveness of current management strategies. The objective of this study was to analyse L. maculans isolates derived from canola stubble in commercial fields collected in 2010 and 2011 across western Canada for the presence and frequency of avirulence (Avr) genes. A total of 674 isolates were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm6, AvrLm7, AvrLm9, AvrLepR1, AvrLepR2 and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR amplification of AvrLm1, AvrLm6 and AvrLm4-Lm7. Certain alleles were more prevalent in the pathogen population, with AvrLm6 and AvrLm7 present in >85% of isolates, while AvrLm3, AvrLm9 and AvrLepR2 were present in <10% of isolates. A total of 55 races (different combinations of Avr alleles) were detected, with the two most common ones being AvrLm2-Lm4-Lm6-Lm7 and AvrLm2-Lm4-Lm6-Lm7-LmS. Races carrying as many as seven and as few as one known Avr allele were detected. Selection pressure from the race-specific resistance genes carried in canola cultivars has probably played a significant role in the current Avr profile, which may have also contributed to the recent increase in blackleg observed in western Canada.

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