Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis.

SignificanceOscillations in intracellular calcium concentration play an essential role in the regulation of multiple cellular processes. In plants capable of root endosymbiosis with nitrogen-fixing bacteria and/or arbuscular mycorrhizal fungi, nuclear localized calcium oscillations are essential to transduce the microbial signal. Although the ion channels required to generate the nuclear localized calcium oscillations have been identified, their mechanisms of regulation are unknown. Here, we combined proteomics and engineering approaches to demonstrate that the calcium-bound form of the calmodulin 2 (CaM2) associates with CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15s), closing the channels and providing the negative feedback to sustain the oscillatory mechanism. We further unraveled that the engineered CaM2 accelerates early endosymbioses and enhanced root nodule symbiosis but not arbuscular mycorrhization.

High frequency of fungicide resistance-associated mutations in the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici.

BACKGROUND: Reliance on fungicides to manage disease creates selection pressure for the evolution of resistance in fungal and oomycete pathogens. Rust fungi (Pucciniales) are major pathogens of cereals and other crops and have been classified as low-risk for developing resistance to fungicides; no case of field failure of fungicides in a cereal rust disease has yet been recorded. Recently, the Asian soybean rust pathogen, Phakopsora pachyrhizi evolved resistance to several fungicide classes, prompting us to screen a large sample of the globally widespread wheat yellow rust pathogen, Puccinia striiformis f. sp. tritici (Pst), for mutations associated with fungicide resistance. RESULTS: We evaluated 363 Pst isolates from Europe, the USA, Ethiopia, Chile, China and New Zealand for mutations in the target genes of demethylase inhibitor (DMI; Cyp51) and succinate dehydrogenase inhibitor (SDHI; SdhB, SdhC and SdhD) fungicides. A high proportion of Pst isolates carrying a Y134F DMI resistance-associated substitution in the Cyp51 gene was found among those from China and New Zealand. A set of geographically diverse Pst isolates was also found to display a substitution in SdhC (I85V) that is homologous to that reported recently in P. pachyrhizi and linked to SDHI resistance. CONCLUSION: The identification of resistance-associated alleles confirms that cereal rusts are not immune to fungicide resistance and that selection for resistance evolution is operating at high levels in certain locations. It highlights the need to adopt fungicide resistance management practices and to monitor cereal rust species for development of resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mutagenesis of Puccinia graminis f. sp. tritici and Selection of Gain-of-Virulence Mutants.

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance (Sr) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr-gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs. To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43, Sr44, or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43, Sr44, or Sr45, respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43. These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance (R) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis.

MARPLE, a point-of-care, strain-level disease diagnostics and surveillance tool for complex fungal pathogens.

BACKGROUND: Effective disease management depends on timely and accurate diagnosis to guide control measures. The capacity to distinguish between individuals in a pathogen population with specific properties such as fungicide resistance, toxin production and virulence profiles is often essential to inform disease management approaches. The genomics revolution has led to technologies that can rapidly produce high-resolution genotypic information to define individual variants of a pathogen species. However, their application to complex fungal pathogens has remained limited due to the frequent inability to culture these pathogens in the absence of their host and their large genome sizes. RESULTS: Here, we describe the development of Mobile And Real-time PLant disEase (MARPLE) diagnostics, a portable, genomics-based, point-of-care approach specifically tailored to identify individual strains of complex fungal plant pathogens. We used targeted sequencing to overcome limitations associated with the size of fungal genomes and their often obligately biotrophic nature. Focusing on the wheat yellow rust pathogen, Puccinia striiformis f.sp. tritici (Pst), we demonstrate that our approach can be used to rapidly define individual strains, assign strains to distinct genetic lineages that have been shown to correlate tightly with their virulence profiles and monitor genes of importance. CONCLUSIONS: MARPLE diagnostics enables rapid identification of individual pathogen strains and has the potential to monitor those with specific properties such as fungicide resistance directly from field-collected infected plant tissue in situ. Generating results within 48 h of field sampling, this new strategy has far-reaching implications for tracking plant health threats.

Ethanol from a biorefinery waste stream: Saccharification of amylase, protease and xylanase treated wheat bran.

Biorefining aims to exploit the full value of plant material by sequentially extracting and valorising its components. Many studies focus on the saccharification of virgin biomass sources, but it may be more efficient to pre-extract high-value components before hydrolysis to fermentable sugars. In the current study, a bran residue from de-starched, protein depleted and xylanase treated wheat bran has been subjected to hydrothermal pretreatment, saccharification and fermentation procedures to convert the residue to ethanol. The most effective pretreatment conditions (>190 °C, 10 min) and saccharification conditions were identified following bench-scale liquid hot water pretreatment. Pre-extraction of enzymatically-hydrolysable starch and xylan reduced the release of furfural production, particularly when lower pretreatment severities were used. Pilot-scale steam explosion of the lignocellulosic residue followed by cellulase treatment and conversion to ethanol at a high substrate concentration (19%) gave an ethanol titre of ≈ 25 g/L or a yield of 93% of the theoretical maximum.