Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

The Changing Virulence of Stripe Rust in Canada from 1984 to 2017.

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, is an important wheat disease worldwide. In this study, the P. striiformis f. sp. tritici population in Canada, representing a time period from 1984 to 2017, was analyzed for virulence diversity and geographical distribution. Virulence of 140 P. striiformis f. sp. tritici isolates was evaluated on 17 near-isogenic wheat lines in the 'Avocet S' background, each containing a single resistance gene along with an 18th line 'Tyee'. Seedlings were inoculated with a urediniospore/talc mixture and infection types were evaluated on a scale of 0 to 9. In total, 89 races were identified with various combinations of defeated Yr genes. Clear changes in pathogen virulence have been observed through time that are confirmed by clustering algorithms. The results showed that the tested P. striiformis f. sp. tritici isolates remained avirulent on Yr1, Yr5, and Yr15, and have very low frequency of virulence on Yr76, but had high frequencies of virulence on Yr6, Yr7, Yr8, Yr9, Yr17, Yr43, Yr44, YrTr1, and YrExp2. P. striiformis f. sp. tritici virulence spiked on Yr7, Yr8, and Yr9 for the first time in 2000, and on Yr10 and Yr27 in 2010. Overall, the predominant races in Canada were very similar to those reported in the United States (PSTv-37, PSTv-41, and PSTv-52), which indicates long-distance migration of P. striiformis f. sp. tritici from the United States to Canada. Sixty-four races had unique virulence combinations that had not been previously reported in the United States, which suggested that evolution of virulence/avirulence for host resistance by mutation at local scale, is possible. Analysis of diversity between Canadian isolates and races from the United States since 2010 showed that the P. striiformis f. sp. tritici population in western Canada is similar to that in the western states of the United States, and that the population in eastern Canada is similar to the eastern and/or central regions of the United States, supporting the hypothesis that specific P. striiformis f. sp. tritici populations in North America travel through different wind trajectories.

Targeted mutagenesis in wheat microspores using CRISPR/Cas9.

CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10-20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.

Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4-7.

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.

Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach.

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.

Genetic mapping of the Leptosphaeria maculans avirulence gene corresponding to the LepR1 resistance gene of Brassica napus.

AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to Brassica LepR1, a plant gene controlling dominant, race-specific resistance to this pathogen. An in vitro cross between the virulent L. maculans isolate, 87-41, and the avirulent isolate, 99-56, was performed in order to map the AvrLepR1 gene. The disease reactions of the 94 of the resulting F(1) progenies were tested on the canola line ddm-12-6s-1, which carries LepR1. There were 44 avirulent progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-12-6s-1 is controlled by a single gene. Tetrad analysis also indicated a 1:1 segregation ratio. The AvrLepR1 gene was positioned on a genetic map of L. maculans relative to 259 sequence-related amplified polymorphism (SRAP) markers, two cloned avirulence genes (AvrLm1 and AvrLm4-7) and the mating type locus (MAT1). The genetic map consisted of 36 linkage groups, ranging in size from 13.1 to 163.7 cM, and spanned a total of 2,076.4 cM. The AvrLepR1 locus was mapped to linkage group 4, in the 13.1 cM interval flanked by the SRAP markers SBG49-110 and FT161-223. The AvrLm4-7 locus was also positioned on linkage group 4, close to but distinct from the AvrLepR1 locus, in the 5.4 cM interval flanked by FT161-223 and P1314-300. This work will make possible the further characterization and map-based cloning of AvrLepR1. A combination of genetic mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence genes that have been characterized previously.

Developing rainfall- and temperature-based models to describe infection of canola under field conditions caused by pycnidiospores of Leptosphaeria maculans.

Blackleg, also known as Phoma stem canker, caused by Leptosphaeria maculans (Phoma lingam), is one of the most serious diseases of canola worldwide. In this study, the mean disease severity (Ds) and incidence (Di) of canola cv. Westar plants infected at the cotyledon, three-leaf, and six-leaf stages by pycnidiospores of L. maculans were monitored in the greenhouse after infection of the plants under field conditions in two successive years and associated with meteorological data during infection time. Pearson's correlation coefficient showed that total rainfall per week (R) was significantly correlated to Ds on plants infected at the cotyledon, three-leaf, and six-leaf stages, and average maximum temperature per week (Tmax) only showed significant correlation with plants infected at the cotyledon and six-leaf stages. These results also indicated that there is correlation between Di and R for plants infected at all three growth stages. A nonlinear model was developed to evaluate the combined effects of R and Tmax on Ds. The best model comprised monomolecular function and beta probability density function for plants infected at the above three growth stages. Parameters, including maximum potential for Ds at a given rainfall (d(max)), rate of changes with respect to rainfall (k), constant of integration (B), maximum potential for Ds with respect to Tmax (e), rate of increase with increasing Tmax to optimum (n), and rate of decrease as Tmax increased and passed the optimum Tmax (p), were estimated for plants infected at the above three growth stages. The effect of plant growth stage was characterized by differences in the upper limit parameter a. This parameter was greater for the plants infected at the cotyledon stage than for plants infected at the other two stages. The estimate of parameter k was the same for the plants infected at the cotyledon and three-leaf stages. This parameter was much lower for the plants infected at the six-leaf stage compared with two other stages. The logistic model could describe the disease incidence with respect to R slightly better than the other two models in the plants infected at all three growth stages. Based on the model, upper-limit estimate (d(max)) was approximately 100, 94.4, and 88.8% in the plants infected at cotyledon, three-leaf, and six-leaf stages, respectively. Di increased until rainfall reached approximately 18, 10, and 13 mm/week and became constant in the plants at cotyledon, three-leaf, and six-leaf stages, respectively. Effects of plant growth stage on the rate of change with respect to R (parameter k) were lower in the plants infected at cotyledon than at the other two stages. The accuracy of the nonlinear models suggests that they could be used to develop a comprehensive model to evaluate epidemics of blackleg based on pycnidiospores as sources of inoculum. However, additional years of data collection should improve model fit and evaluation of introduced models and contribute to the development of a more robust predictive model.