A genome of Pyricularia oryzae (synonym Magnaporthe oryzae), the fungus that causes blast disease on diverse grass species, has seven core chromosomes and may contain supernumerary mini-chromosomes. The P. oryzae Triticum (PoT) pathotype is the phylogenetic lineage responsible for devastating epidemics of wheat blast disease. Genomic analysis of wheat blast field isolates from the initial outbreak in 1985 in Brazil through recent field isolates in South America revealed dynamic presence and structure of mini-chromosomes. Two earliest field isolates representing founder lineages for the Triticum pathotype contain similar mini-chromosomes. Another PoT founder isolate from 1986 and 37 out of 39 Triticum field isolates collected between 1986 and 1992 lack mini-chromosomes. Mini-chromosomes present in the founder strains each contain two copies of the PWT7 wheat blast avirulence gene, and PWT7 was lost from subsequent early strains through mini-chromosome loss. Almost all PoT field isolates from 2005 to 2020 have regained mini-chromosomes in which PWT7 sequences have been replaced by other sequences. Telomere-to-telomere assemblies of 11 mini-chromosomes identified two major mini-chromosome types in the South American PoT population, and demonstrated significant within-mini-chromosome sequence alterations as well as recombination with other mini-chromosomes or core chromosome ends. Additionally, our data indicate horizontal mini-chromosome transfer between Pyricularia species, resulting in nearly identical genomic fragments shared between P. oryzae and Pyricularia pennisetigena isolates in the PWT4 avirulence gene region. Our genomic analysis depicts the dynamic mini-chromosome compartment in the diverse South American Triticum field population through time, indicating important roles for mini-chromosomes in pathogen adaptation and pathogenicity.

Fungal pathogens display remarkable variation in genome content and organization that directly impacts their survival and host interactions. Although numerous models have been proposed to explain the origins of this variation, they generally fail to explain or predict the mechanisms that generate the genome variation observed in natural populations. Starships are a recently discovered group of giant fungal transposons that carry dozens of genes as cargo and horizontally transfer both within and between species. Here, we identify the features of a newly defined ' Starship compartment' in the major fungal plant pathogen Pyricularia oryzae . We test the hypothesis that the Starship compartment makes distinct contributions to fungal genome evolution by explicitly comparing its transferability, mutability, and epigenetic modifications with those of the canonical core and accessory compartments. To enable this, we developed an updated and user-friendly version of the annotation tool stargraph for the comprehensive annotation of Starships and Starship -like regions. Using this approach, we identified two distinct families of Starships and related Starship -like regions in P. oryzae that differ in their activity, impacts on genome organization, modes of sequence evolution, and epigenetic modifications. Elements from the more active family exhibit higher rates of structural variation than all other genomic compartments in the predominantly clonal isolates infecting rice. Both families of Starships encode specific suites of known effector sequences that contribute to plant disease and Starship activity accounts for avirulence gene turnover, which suggests that evolutionary change within the Starship compartment may subsequently impact the evolution of plant-fungal interactions. Starships from the more active family have repeatedly transferred across the Pyricularia genus and tend to be depleted in heterochromatic histone modifications and repeat-induced point mutations. However, contrasting histone modification profiles in this family suggests a genomic conflict between silencing or maintaining Starship activity. Our findings demonstrate that variation in the mode of sequence diversification and epigenetic modification within the Starship compartment underpins the impacts of these giant transposons on fungal genome evolution. We argue for the explicit consideration of not only the Starship compartment but of element-specific dynamics when investigating the evolution of host-fungal interactions. ### Competing Interest Statement The authors have declared no competing interest. United States Department of Agriculture, https://ror.org/01na82s61, 2021-67013-35724, 2021-68013-33719 U.S. National Science Foundation, https://ror.org/021nxhr62, 2311738, 2448034, 2011500

Magnaporthe oryzae (syn. Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. Individual M. oryzae strains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of a Lolium strain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Contour-clamped homogeneous electric field (CHEF) karyotyping and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Optimization of a fluorescence in situ hybridization (FISH) protocol enabled single-cell karyotyping and revelation of drastic cellular variation in numbers of mini-chromosomes. In addition, rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. We found that, in contrast to Long Terminal Repeat (LTR) retrotransposons in core chromosomes, LTR retrotransposons in mini-chromosomes were more recently inserted, less methylated, and with higher G + C content. The data indicated that most LTR retrotransposons in mini-chromosomes retain high activity and have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation, which may contribute to highly dynamic mini-chromosome content in fungi.

Abstract Magnaporthe oryzae(syn.Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. IndividualM.oryzaestrains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of aLoliumstrain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Karyotyping analysis and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. The susceptibility to structural variation in mini-chromosomes may be associated with repetitive features and the high activity of transposable elements in mini-chromosomes, in which many intact retrotransposons were recently inserted, largely unmethylated, and likely have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation.

Magnaporthe oryzae (syn. Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. Individual M. oryzae strains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of a Lolium strain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Karyotyping analysis and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. The susceptibility to structural variation in mini-chromosomes may be associated with repetitive features and the high activity of transposable elements in mini-chromosomes, in which many intact retrotransposons were recently inserted, largely unmethylated, and likely have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation. ### Competing Interest Statement SL is the co-founder of Data2Bio, LLC. Other authors claim no competing interest.

The genomes of the fungus Magnaporthe oryzae that causes blast diseases on diverse grass species, including major crops, have indispensable core-chromosomes and may contain supernumerary chromosomes, also known as mini-chromosomes. These mini-chromosomes are speculated to provide effector gene mobility, and may transfer between strains. To understand the biology of mini-chromosomes, it is valuable to be able to detect whether a M. oryzae strain possesses a mini-chromosome. Here, we applied recurrent neural network models for classifying DNA sequences as arising from core- or mini-chromosomes. The models were trained with sequences from available core- and mini-chromosome assemblies, and then used to predict the presence of mini-chromosomes in a global collection of M. oryzae isolates using short-read DNA sequences. The model predicted that mini-chromosomes were prevalent in M. oryzae isolates. Interestingly, at least one mini-chromosome was present in all recent wheat isolates, but no mini-chromosomes were found in early isolates collected before 1991, indicating a preferential selection for strains carrying mini-chromosomes in recent years. The model was also used to identify assembled contigs derived from mini-chromosomes. In summary, our study has developed a reliable method for categorizing DNA sequences and showcases an application of recurrent neural networks in predictive genomics.

The fungal pathogen, Magnaporthe oryzae Triticum pathotype, causing wheat blast disease was first identified in South America and recently spread across continents to South Asia and Africa. Here, we studied the genetic relationship among isolates found on the three continents.Magnaporthe oryzae strains closely related to a South American field isolate B71 were found to have caused the wheat blast outbreaks in South Asia and Africa. Genomic variation among isolates from the three continents was examined using an improved B71 reference genome and whole-genome sequences. We found strong evidence to support that the outbreaks in Bangladesh and Zambia were caused by the introductions of genetically separated isolates, although they were all close to B71 and, therefore, collectively referred to as the B71 branch. In addition, B71 branch strains carried at least one supernumerary mini-chromosome. Genome assembly of a Zambian strain revealed that its mini-chromosome was similar to the B71 mini-chromosome but with a high level of structural variation. Our findings show that while core genomes of the multiple introductions are highly similar, the mini-chromosomes have undergone marked diversification. The maintenance of the mini-chromosome and rapid genomic changes suggest the mini-chromosomes may serve important virulence or niche adaptation roles under diverse environmental conditions.

Cellular biological networks represent the molecular interactions that shape function of living cells. Uncovering the organization of a biological network requires efficient and accurate algorithms to determine the components, termed communities, underlying specific processes. Detecting functional communities is challenging because reconstructed biological networks are always incomplete due to technical bias and biological complexity, and the evaluation of putative communities is further complicated by a lack of known ground truth. To address these challenges, we developed a geometric-based detection framework based on Ollivier-Ricci curvature to exploit information about network topology to perform community detection from partially observed biological networks. We further improved this approach by integrating knowledge of gene function, termed side information, into the Ollivier-Ricci curvature algorithm to aid in community detection. This approach identified essential conserved and varied biological communities from partially observed Arabidopsis protein interaction datasets better than the previously used methods. We show that Ollivier-Ricci curvature with side information identified an expanded auxin community to include an important protein stability complex, the Cop9 signalosome, consistent with previous reported links to auxin response and root development. The results show that community detection based on Ollivier-Ricci curvature with side information can uncover novel components and novel communities in biological networks, providing novel insight into the organization and function of complex networks.

Background RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. Results Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. Conclusions Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.

Ralstonia solanacearum thrives in plant xylem vessels and causes bacterial wilt disease despite the low nutrient content of xylem sap. We found that R. solanacearum manipulates its host to increase nutrients in tomato xylem sap, enabling it to grow better in sap from infected plants than in sap from healthy plants. Untargeted GC/MS metabolomics identified 22 metabolites enriched in R. solanacearum-infected sap. Eight of these could serve as sole carbon or nitrogen sources for R. solanacearum. Putrescine, a polyamine that is not a sole carbon or nitrogen source for R. solanacearum, was enriched 76-fold to 37 µM in R. solanacearum-infected sap. R. solanacearum synthesized putrescine via a SpeC ornithine decarboxylase. A ΔspeC mutant required ≥ 15 µM exogenous putrescine to grow and could not grow alone in xylem even when plants were treated with putrescine. However, co-inoculation with wildtype rescued ΔspeC growth, indicating R. solanacearum produced and exported putrescine to xylem sap. Intriguingly, treating plants with putrescine before inoculation accelerated wilt symptom development and R. solanacearum growth and systemic spread. Xylem putrescine concentration was unchanged in putrescine-treated plants, so the exogenous putrescine likely accelerated disease indirectly by affecting host physiology. These results indicate that putrescine is a pathogen-produced virulence metabolite.

BACKGROUND:Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes.RESULTS:We report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development.CONCLUSION:The nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.