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The MD-2-related lipid-recognition (ML/Md-2) domain is a lipid/sterol-binding domain that are involved in sterol transfer and innate immunity in eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 30 fungi including plant pathogens. All the studied species were found to have varied ML numbers, and expansion of the family was observed in Rhizophagus irregularis (RI) with 33 genes. The molecular docking studies of these proteins with cholesterol derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. The phylogenetic studies among eukaryotic ML proteins showed that Puccinia ML members are more closely associated with animal (insect) npc2 proteins than other fungal ML members. One of the candidates from leaf rust fungus Puccinia triticina, Pt5643 was PCR amplified and further characterized using various studies such as qRT-PCR, subcellular localization studies, yeast functional complementation, signal peptide validation, and expression studies. The Pt5643 exhibits the highest expression on the 5th day post-infection (dpi). The confocal microscopy of Pt5643 in onion epidermal cells and N. benthamiana shows its location in the cytoplasm and nucleus. The functional complementation studies of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic/yeast npc2 gene. Furthermore, the overexpression of Pt5643 also suppressed the BAX, NEP1, and H₂O₂-induced program cell death in Nicotiana species and yeast. Altogether the present study reports the novel function of ML domain proteins in plant fungal pathogens and their possible role as effector molecules in host defense manipulation., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: Stem rust resistance was mainly based on a few, already known resistance genes; for yellow rust resistance there was a combination of designated genes and minor QTLs. Yellow rust (YR) caused by Puccinia striiformis f. sp. tritici (Pst) and stem rust (SR) caused by Puccinia graminis f. sp. tritici (Pgt) are among the most damaging wheat diseases. Although, yellow rust has occurred regularly in Europe since the advent of the Warrior race in 2011, damaging stem rust epidemics are still unusual. We analyzed the resistance of seven segregating populations at the adult growth stage with the parents being selected for YR and SR resistances across three to six environments (location-year combinations) following inoculation with defined Pst and Pgt races. In total, 600 progenies were phenotyped and 563 were genotyped with a 25k SNP array. For SR resistance, three major resistance genes (Sr24, Sr31, Sr38/Yr17) were detected in different combinations. Additional QTLs provided much smaller effects except for a gene on chromosome 4B that explained much of the genetic variance. For YR resistance, ten loci with highly varying percentages of explained genetic variance (pG, 6-99%) were mapped. Our results imply that introgression of new SR resistances will be necessary for breeding future rust resistant cultivars, whereas YR resistance can be achieved by genomic selection of many of the detected QTLs., (© 2024. The Author(s).)

Wheat leaf rust, caused by the fungus Puccinia triticina (Pt), severely affects the grain quality and quantity of bread wheat (Triticum aestivum L.). Hairpin small(s)RNAs, like micro(mi)RNAs and their variants [including isomiRNAs (isomiRs) and microRNA-like RNAs (milRNAs)], along with their corresponding target genes, bestow leaf rust disease resistance, development and progression from both interacting species. However, the regulatory networks remain inadequately understood. Thirteen differentially expressed novel miRNAs, including two isomiRs and three milRNAs were discerned from induced reads of wheat sRNA libraries, and a further 5,393 and 1,275 candidate target genes were predicted in wheat and Pt, respectively. Functional annotation divulged that wheat-originated miRNAs/isomiRs were involved in resistance, while Pt-derived milRNAs imparted pathogenesis. The identified milRNAs- Tae-Pt-milR5, Tae-Pt-milR12, and Tae-Pt-milR14b and their cleavage sites on Pt target gene MEP5 were confirmed through degradome library screening, suggesting cross-kingdom translocation of Pt virulent genes in wheat host. Co-expression analysis of miRNAs/isomiRs-target genes provided insights into combating leaf rust disease, while co-expression analysis of milRNAs-target gene pairs reflected the extent of pathogenicity exerted by Pt with varied expression levels at the analyzed time points. The analysis pinpointed leaf rust-responsive candidate hairpin sRNAs- Tae-miR8, Tae-Pt-miR12, Tae-Pt-miR14a, and Tae-Pt-miR14b in wheat and Tae-Pt-milR12 in Pt. This study provides new insights into the hairpin sRNAs involved in the resistance and pathogenesis of wheat and Pt, respectively. Furthermore, crucial hairpin sRNAs and their promising targets for future biotechnological interventions to augment stress resilience have been identified., (© 2024 Scandinavian Plant Physiology Society.)

The acquisition of nutrients from host plants by phytopathogenic fungi is critically important for their invasion success. Melampsora larici-populina, an obligate biotrophic pathogenic fungus, causes the poplar leaf rust disease and can severely damage host poplar plants. Previously, we found that oligopeptide transporters (OPTs) have undergone a convergent expansion, which might reflect adaptation to a phytoparasitic lifestyle. Here, we used various methods to evaluate this hypothesis, including conserved motif identification, positive selection signal mining, expression pattern clustering analysis, and neutral selection tests. The motif composition of the five clades in the OPT family differed, and positive selection was observed during clade differentiation. This suggests that OPTs in these five clades may be functionally differentiated, which would increase the range of transported substrates and promote the absorption of more types of nitrogen compounds from the hosts. According to clustering analysis of gene expression patterns, the expression of most genes from the two expanded clades (clade 2 and 4) was up-regulated during the infection of poplar trees, indicating that the expansion of OPTs likely occurred to promote the uptake of oligopeptides from host poplar plants. The MellpOPT4g gene was determined to be under significant balancing selection based on the neutral selection tests, suggesting that it plays a role in the pathogenic process. In conclusion, these three observations provide preliminary evidence supporting our hypothesis, as they indicate that the expansion of OPTs in M. larici-populina has aided the ability of this pathogen to acquire nutrients from host plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Puccinia graminis f. sp. tritici (Pgt) Ug99-emerging virulent races present a major challenge to global wheat production. To meet present and future needs, new sources of resistance must be found. Identification of markers that allow tracking of resistance genes is needed for deployment strategies to combat highly virulent pathogen races. Field evaluation of a DH population located a QTL for stem rust (Sr) resistance, QSr.nc-6D from the breeding line MD01W28-08-11 to the distal region of chromosome arm 6DS where Sr resistance genes Sr42, SrCad, and SrTmp have been identified. A locus for seedling resistance to Pgt race TTKSK was identified in a DH population and an RIL population derived from the cross AGS2000 × LA95135. The resistant cultivar AGS2000 is in the pedigree of MD01W28-08-11 and our results suggest that it is the source of Sr resistance in this breeding line. We exploited published markers and exome capture data to enrich marker density in a 10 Mb region flanking QSr.nc-6D. Our fine mapping in heterozygous inbred families identified three markers co-segregating with resistance and delimited QSr.nc-6D to a 1.3 Mb region. We further exploited information from other genome assemblies and identified collinear regions of 6DS harboring clusters of NLR genes. Evaluation of KASP assays corresponding to our co-segregating SNP suggests that they can be used to track this Sr resistance in breeding programs. However, our results also underscore the challenges posed in identifying genes underlying resistance in such complex regions in the absence of genome sequence from the resistant genotypes., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The common wheat line 4N0461 showed adult-plant resistance to leaf rust. 4N0461 was crossed with susceptible cultivars Nongda4503 and Shi4185 to map the causal resistance gene(s). Segregation of leaf rust response in F 2 populations from both crosses was 9 resistant:7 susceptible, indicative of two complementary dominant resistance genes. The genes were located on chromosome arms 3BS and 4BL and temporarily named LrN3B and LrN4B, respectively. Subpopulations from 4N0461 × Nongda4503 with LrN3B segregating as a single allele were used to fine-map LrN3B locus. LrN3B was delineated in a genetic interval of 0.07 cM, corresponding to 106 kb based on the Chinese Spring reference genome (IWGSC RefSeq v1.1). Four genes were annotated in this region, among which TraesCS3B02G014800 and TraesCS3B02G014900 differed between resistant and susceptible genotypes, and both were required for LrN3B resistance in virus-induced gene silencing experiments. Diagnostic markers developed for checking the polymorphism of each candidate gene, can be used for marker-assisted selection in wheat breeding programs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: A new stripe rust resistance gene YrBDT in Chinese landrace wheat Baidatou was mapped to a 943.6-kb interval on chromosome arm 6DS and co-segregated with a marker CAPS3 developed from candidate gene TraesCS6D03G0027300. Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease of wheat. Chinese landrace wheat Baidatou has shown high resistance to a broad spectrum of Pst races at both the seedling and adult-plant stages for decades in the Longnan region of Gansu province, a hot spot for stripe rust epidemics. Here, we report fine mapping and candidate gene analysis of stripe rust resistance gene YrBDT in Baidatou. Analysis of F 1 , F 2 plants and F 2:3 lines indicated that resistance in Baidatou to Pst race CYR31 was conferred by a single dominant gene, temporarily designated YrBDT. Bulked segregant exome capture sequencing (BSE-seq) analysis revealed 61 high-confidence polymorphic SNPs concentrated in a 5.4-Mb interval at the distal of chromosome arm 6DS. Several SNPs and InDels were also identified by genome mining of DNA sampled from the parents and contrasting bulks. The YrBDT locus was mapped to a 943.6-kb (4,658,322-5,601,880 bp) genomic region spanned by markers STS2 and STS3 based on IWGSC RefSeq v2.1, including five putative disease resistance genes. There was high collinearity of the target interval among Chinese Spring RefSeq v2.1, Ae. tauschii AL8/78 and Fielder genomes. The expression level of TraesCS6D03G0027300 showed significant association with Pst infection, and a gene-specific marker CAPS3 developed from TraesCS6D03G0027300 co-segregated with YrBDT suggesting this gene as a candidate of YrBDT. The resistance gene and flanking markers can be used in marker-assisted selection for improvement of stripe rust resistance., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Wheat yellow (stripe) rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most devastating diseases of wheat worldwide. Pst populations are composed of multiple genetic groups, each carrying one or more races characterized by different avirulence/virulence combinations. Since the severe epidemics in 2017, yellow rust has become the most economically important wheat foliar disease in Uruguay. A set of 124 Pst isolates collected from wheat fields in Uruguay between 2017 and 2021 were characterized phenotypically, and 27 of those isolates were subsequently investigated in-depth by additional molecular genotyping and race phenotyping analyses. Three genetic groups were identified, PstS7 , PstS10 , and PstS13 , with the latter being the most prevalent. Two races previously reported in Europe, Warrior ( PstS7 ) and Benchmark ( Pst S10), were detected in four and two isolates, respectively. A third race, known as Triticale2015 ( PstS13 ), that was first detected in Europe in 2015 and in Argentina in 2017 was detected at several locations. Additional virulence to Yr3 , Yr17 , Yr25 , Yr27 , or Yr32 was detected in three new race variants within PstS13 . The identification of these new races, which have not been reported outside South America, provides strong evidence of the local evolution of virulence in Pst during the recent epidemic years., Competing Interests: The author(s) declare no conflict of interest.

Stem rust, caused by the biotrophic fungal pathogen Puccinia graminis f. sp. tritici ( Pgt ), is an important disease of wheat. However, the majority of Pgt virulence/avirulence loci and underlying genes remain uncharacterized due to the constraints of developing bi-parental populations with this obligate biotroph. Genome-wide association studies (GWAS) using a sexual Pgt population mainly collected from the Pacific Northwestern United States were used to identify candidate virulence/avirulence effector genes corresponding to the six wheat Sr genes: Sr5 , Sr21 , Sr8a , Sr17 , Sr9a , and Sr9d . The Pgt isolates were genotyped using whole-genome shotgun sequencing that identified approximately 1.2 million single nucleotide polymorphisms (SNPs) and were phenotyped at the seedling stage on six Sr gene differential lines. Association mapping analyses identified 17 Pgt loci associated with virulence or avirulence phenotypes on six Pgt resistance genes. Among these loci, 16 interacted with a specific Sr gene, indicating Sr -gene specific interactions. However, one avirulence locus interacted with two separate Sr genes ( Sr9a and Sr17 ), suggesting two distinct Sr genes identifying a single avirulence effector. A total of 24 unique effector gene candidates were identified, and haplotype analysis suggests that within this population, AvrSr5 , AvrSr21 , AvrSr8a , AvrSr17 , and AvrSr9a are dominant avirulence genes, while avrSr9d is a dominant virulence gene. The putative effector genes will be fundamental for future effector gene cloning efforts, allowing for further understanding of rust effector biology and the mechanisms underlying virulence evolution in Pgt with respect to race-specific R -genes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Tea ( Camellia sinensis [L.] O. Kuntze) is cultivated as a beverage crop. Despite being a hardy perennial, the tea plant is susceptible to various biotic stresses. Among them, the foliar disease blister blight (BB) is considered the most serious threat to the tea industry, particularly in Asia. BB caused by Exobasidium vexans (Basidiomycetes) was first reported from Northern India in 1868 and gradually established in other tea-growing countries. The fungus E. vexans attacks young harvestable shoots and causes 20 to 50% crop loss. Over the past 150 years, scientific research has delved into various aspects of BB disease, including pathogen biology, disease cycle, epidemiology, disease forecasting, crop loss assessment, and disease management strategies. In a recent shift in research focus, scientists have begun to investigate the resistance mechanisms of tea plants against BB and apply this knowledge to commercial tea cultivation. Although progress has been significant in understanding the fundamental aspects of BB resistance, the detailed molecular mechanisms driving this resistance remain under investigation. This paper focuses on the current understanding of defense mechanisms employed by tea plants against E. vexans and, conversely, how E. vexans overcomes these defenses. Furthermore, we discuss the application of plant resistance strategies in commercial tea cultivation. Lastly, we identify existing research gaps and propose future research directions in the field., Competing Interests: The author(s) declare no conflict of interest.

Tilletia indica Mitra causes Karnal bunt (KB) in wheat by pathogenic dikaryophase. The present study is the first to provide the draft genomes of the dikaryon (PSWKBGD-3) and its two monosporidial lines (PSWKBGH-1 and 2) using Illumina and PacBio reads, their annotation and the comparative analyses among the three genomes by extracting polymorphic SSR markers. The trancriptome from infected wheat grains of the susceptible wheat cultivar WL711 at 24 h, 48h, and 7d after inoculation of PSWKBGH-1, 2 and PSWKBGD-3 were also isolated. Further, two transcriptome analyses were performed utilizing T. indica transcriptome to extract dikaryon genes responsible for pathogenesis, and wheat transcriptome to extract wheat genes affected by dikaryon involved in plant-pathogen interaction during progression of KB in wheat. A total of 54, 529, and 87 genes at 24hai, 48hai, and 7dai, respectively were upregulated in dikaryon stage while 21, 35, and 134 genes of T. indica at 24hai, 48hai, and 7dai, respectively, were activated only in dikaryon stage. While, a total of 23, 17, and 52 wheat genes at 24hai, 48hai, and 7dai, respectively were upregulated due to the presence of dikaryon stage only. The results obtained during this study have been compiled in a web resource called TiGeR ( http://backlin.cabgrid.res.in/tiger/ ), which is the first genomic resource for T. indica cataloguing genes, genomic and polymorphic SSRs of the three T. indica lines, wheat and T. indica DEGs as well as wheat genes affected by T. indica dikaryon along with the pathogenecity related proteins of T. indica dikaryon during incidence of KB at different time points. The present study would be helpful to understand the role of dikaryon in plant-pathogen interaction during progression of KB, which would be helpful to manage KB in wheat, and to develop KB-resistant wheat varieties., (© 2024. The Author(s).)

Wheat is an essential food commodity cultivated throughout the world. However, this crop faces continuous threats from fungal pathogens, leaf rust (LR) and stripe rust (YR). To continue feeding the growing population, these major destructors of wheat must be effectively countered by enhancing the genetic diversity of cultivated germplasm. In this study, an introgression line with hexaploid background (IL sp3603 ) carrying resistance against Pt pathotypes 77-5 (121R63-1), 77-9 (121R60-1) and Pst pathotypes 46S119 (46E159), 110S119 (110E159), 238S119 (238E159) was developed from donor wheat wild progenitor, Aegilops speltoides acc pau 3603. To understand the genetic basis of resistance and map these genes (named Lr sp3603 and Yr sp3603 ), inheritance studies were carried out in F 6 and F 7 mapping population, developed by crossing IL sp3603 with LR and YR susceptible cultivar WL711, which revealed a monogenic (single gene) inheritance pattern for each of these traits. Bulk segregant analysis combined with 35 K Axiom SNP array genotyping mapped both genes as separate entities on the short arm of chromosome 6B. A genetic linkage map, comprising five markers, 1 SNP, 1 PLUG and three gene based SSRs, covered a genetic distance of 12.65 cM. Lr sp3603 was flanked by markers Tag-SSR14 (located proximally at 2.42 cM) and SNP AX-94542331 (at 3.28 cM) while Yr sp3603 was mapped at one end closest to AX-94542331 at 6.62 cM distance. Functional annotation of Lr sp3603 target region (∼ 1 Mbp) revealed 10 gene IDs associated with disease resistance mechanisms including three encoding typical R gene domains., (© 2024. The Author(s).)

Background: JUB1, a NAC domain containing hydrogen peroxide-induced transcription factor, plays a critical role in plant immunity. Little is known about how JUB1 responds to leaf rust disease in wheat. Recent discoveries in genomics have also unveiled a multitude of sORFs often assumed to be non-functional, to argue for the necessity of including them as potential regulatory players of translation. However, whether methylation on sORFs spanning the 3'UTR of regulatory genes like JUB1 modulate gene expression, remains unclear., Methods and Results: In this study, we identified the methylation states of two sORFs in 3'UTR of a homologous gene of JUB1 in wheat, TaJUB1-L, at cytosine residues in CpG, CHH and CHG sites at different time points of disease progression in two near-isogenic lines of wheat (HD2329), with and without Lr24 gene during leaf rust pathogenesis. Here, we report a significant demethylation of the CpG dinucleotides occurring in the sORFs of the 3'UTR in the resistant isolines after 24 h post-infection. Also, the up-regulated gene expression observed through RT-qPCR was directly proportional to the demethylation of the CpG sites in the sORFs., Conclusions: Our findings indicate that TaJUB1-L might be a positive regulator in providing tolerance during leaf rust pathogenesis and cytosine methylation at 3'UTR might act as a switch for its expression control. These results enrich the potential benefit of conventional methylation assay techniques for unraveling the unexplored enigma in epigenetics during plant-pathogen interaction in a cost-effective and confidentially conclusive manner., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Rust diseases, including leaf rust, stripe/yellow rust, and stem rust, significantly impact wheat (Triticum aestivum L.) yields, causing substantial economic losses every year. Breeding and deployment of cultivars with genetic resistance is the most effective and sustainable approach to control these diseases. The genetic toolkit for wheat breeders to select for rust resistance has rapidly expanded with a multitude of genetic loci identified using the latest advances in genomics, mapping and cloning strategies. The goal of this review was to establish a wheat genome atlas that provides a comprehensive summary of reported loci associated with rust resistance. Our atlas provides a summary of mapped quantitative trait loci (QTL) and characterised genes for the three rusts from 170 publications over the past two decades. A total of 920 QTL or resistance genes were positioned across the 21 chromosomes of wheat based on the latest wheat reference genome (IWGSC RefSeq v2.1). Interestingly, 26 genomic regions contained multiple rust loci suggesting they could have pleiotropic effects on two or more rust diseases. We discuss a range of strategies to exploit this wealth of genetic information to efficiently utilise sources of resistance, including genomic information to stack desirable and multiple QTL to develop wheat cultivars with enhanced resistance to rust disease., (© 2024. The Author(s).)

Puccinia coronata f. sp. avenae ( Pca ) is an important foliar pathogen of oat which causes crown rust disease. The virulence profile of 48 Pca isolates derived from different locations in Australia was characterized using a collection of oat lines often utilized in rust surveys in the United States and Australia. This analysis indicates that Pca populations in Eastern Australia are broadly virulent, which contrasts with the population in Western Australia (WA). Several oat lines/ Pc genes are effective against all rust samples collected from WA, suggesting they may provide useful resistance in this region if deployed in combination. We identified 19 lines from the United States oat differential set that display disease resistance to Pca in WA, with some in agreement with previous rust survey reports. We adopted the 10-letter nomenclature system to define oat crown rust races in Australia and compare the frequency of those virulence traits to published data from the United States. Based on this nomenclature, 42 unique races were detected among the 48 isolates, reflecting the high diversity of virulence phenotypes for Pca in Australia. Nevertheless, the Pca population in the United States is substantially more broadly virulent than that of Australia. Close examination of resistance profiles for the oat differential set lines after infection with Pca supports hypotheses of allelism or redundancy among Pc genes or the presence of several resistance genes in some oat differential lines. These findings illustrate the need to deconvolute the oat differential set using molecular tools.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Employing race-specific resistance genes remains an effective strategy to protect wheat from leaf rust caused by Puccinia triticina (Pt) worldwide, while the newly emerged Pt races, owing to rapid genetic evolution, frequently overcome the immune response delivered by race-specific resistance genes. The molecular mechanisms underlying the newly evolved virulence Pt pathogen remain unknown. Here, we identified an avirulence protein AvrLr15 from Pt that induced Lr15-dependent immune responses. Heterologously produced AvrLr15 triggered pronounced cell death in Lr15-isogenic wheat leaves. AvrLr15 contains a functional signal peptide, localized to the plant nucleus and cytosol and can suppress BAX-induced cell death. Evasion of Lr15-mediated resistance in wheat was associated with a deletion and point mutations of amino acids in AvrLr15 rather than AvrLr15 gene loss in the Lr15-breaking Pt races, implying that AvrLr15 is required for the virulence function of Pt. Our findings identified the first molecular determinant of wheat race-specific immunity and facilitated the identification of the first AVR/R gene pair in the Pt-wheat pathosystem, which will provide a molecular marker to monitor natural Pt populations and guide the deployment of Lr15-resistant wheat cultivars in the field., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Ten years ago, (black) stem rust - the most damaging of wheat (Triticum aestivum) rusts - re-emerged in western Europe. Disease incidences have since increased in scale and frequency. Here, we investigated the likely underlying causes and used those to propose urgently needed mitigating actions. We report that the first large-scale UK outbreak of the wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt), in 2022 may have been caused by timely arrival of airborne urediniospores from southwest Europe. The drive towards later-maturing wheat varieties in the UK may be exacerbating Pgt incidences, which could have disastrous consequences. Indeed, infection assays showed that two UK Pgt isolates from 2022 could infect over 96% of current UK wheat varieties. We determined that the temperature response data in current disease risk simulation models are outdated. Analysis of germination rates for three current UK Pgt isolates showed substantial variation in temperature response functions, suggesting that the accuracy of disease risk simulations would be substantially enhanced by incorporating data from prevailing Pgt isolates. As Pgt incidences continue to accelerate in western Europe, we advocate for urgent action to curtail Pgt losses and help safeguard future wheat production across the region., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Wheat stem rust, which is caused by Puccinia graminis f. sp. tritici ( Pgt ), is a highly destructive disease that affects wheat crops on a global scale. In this study, the reactions of 150 bread wheat varieties were evaluated for natural Pgt infection at the adult-plant stage in the 2019-2020 and 2020-2021 growing seasons, and they were analyzed using specific molecular markers to detect stem rust resistance genes ( Sr22 , Sr24 , Sr25 , Sr26 , Sr31 , Sr38 , Sr50 , and Sr57 ). Based on phenotypic data, the majority of the varieties (62%) were resistant or moderately resistant to natural Pgt infection. According to molecular results, it was identified that Sr57 was present in 103 varieties, Sr50 in nine varieties, Sr25 in six varieties, and Sr22 , Sr31 , and Sr38 in one variety each. Additionally, their combinations Sr25 + Sr50 , Sr31 + Sr57 , Sr38 + Sr50 , and Sr38 + Sr57 were detected in these varieties. On the other hand, Sr24 and Sr26 were not identified. In addition, many varieties had low stem rust scores, including a large minority that lacked Sr57 . These varieties must have useful resistance to stem rust and could be the basis for selecting greater, possibly durable resistance., Competing Interests: The author declares that they have no competing interests., (© 2024 Cat.)

Fungal diseases, such as powdery mildew and rusts, significantly affect the quality and yield of wheat. Pyramiding diverse types of resistance genes into cultivars represents the preferred strategy to combat these diseases. Moreover, achieving collaborative improvement between diseases resistance, abiotic stress, quality, and agronomic and yield traits is difficult in genetic breeding. In this study, the wheat cultivar, Guinong 29 (GN29), showed high resistance to powdery mildew and stripe rust at both seedling and adult plant stages, and was susceptible to leaf rust at the seedling stage but slow resistance at the adult-plant stage. Meanwhile, it has elite agronomic and yield traits, indicating promising coordination ability among multiple diseases resistance and other key breeding traits. To determine the genetic basis of these elite traits, GN29 was tested with 113 molecular markers for 98 genes associated with diseases resistance, stress tolerance, quality, and adaptability. The results indicated that two powdery mildew resistance (Pm) genes, Pm2 and Pm21, confirmed the outstanding resistance to powdery mildew through genetic analysis, marker detection, genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), and homology-based cloning; the stripe rust resistance (Yr) gene Yr26 and leaf rust resistance (Lr) genes Lr1 and Lr46 conferred the stripe rust and slow leaf rust resistance in GN29, respectively. Meanwhile, GN29 carries dwarfing genes Rht-B1b and Rht-D1a, vernalization genes vrn-A1, vrn-B1, vrn-D1, and vrn-B3, which were consistent with the phenotypic traits in dwarf characteristic and semi-winter property; carries genes Dreb1 and Ta-CRT for stress tolerance to drought, salinity, low temperature, and abscisic acid (ABA), suggesting that GN29 may also have elite stress-tolerance ability; and carries two low-molecular-weight glutenin subunit genes Glu-B3b and Glu-B3bef which contributed to high baking quality. This study not only elucidated the genetic basis of the elite traits in GN29 but also verified the capability for harmonious improvement in both multiple diseases resistance and other comprehensive traits, offering valuable information for breeding breakthrough-resistant cultivars., (© 2024. The Author(s).)

Key Message: The durable stripe rust resistance gene Yr30 was fine-mapped to a 610-kb region in which five candidate genes were identified by expression analysis and sequence polymorphisms. The emergence of genetically diverse and more aggressive races of Puccinia striiformis f. sp. tritici (Pst) in the past twenty years has resulted in global stripe rust outbreaks and the rapid breakdown of resistance genes. Yr30 is an adult plant resistance (APR) gene with broad-spectrum effectiveness and its durability. Here, we fine-mapped the YR30 locus to a 0.52-cM interval using 1629 individuals derived from residual heterozygous F 5:6 plants in a Yaco"S"/Mingxian169 recombinant inbred line population. This interval corresponded to a 610-kb region in the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq version 2.1 on chromosome arm 3BS harboring 30 high-confidence genes. Five genes were identified as candidate genes based on functional annotation, expression analysis by RNA-seq and sequence polymorphisms between cultivars with and without Yr30 based on resequencing. Haplotype analysis of the target region identified six haplotypes (YR30_h1-YR30_h6) in a panel of 1215 wheat accessions based on the 660K feature genotyping array. Lines with YR30_h6 displayed more resistance to stripe rust than the other five haplotypes. Near-isogenic lines (NILs) with Yr30 showed a 32.94% higher grain yield than susceptible counterparts when grown in a stripe rust nursery, whereas there was no difference in grain yield under rust-free conditions. These results lay a foundation for map-based cloning Yr30., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Groundnut is vulnerable to the major foliar fungal disease viz., late leaf spot (LLS) and rust in kharif season, which results in severe yield losses. Until now, LLS and rust resistance linked markers were developed based on GPBD 4 as a major donor source and were validated in its derivatives only, which restricted their use in marker assisted selection (MAS) involving other donors., Methods and Results: The current study focused to validate LLS and rust resistance linked markers employing advanced breeding lines of F 6 generation, derived from nine different crosses involving nine diverse parents, to identify potential markers for marker-assisted breeding of LLS and rust resistance in groundnut. Out of 28-trait linked markers used for validation, 8 were polymorphic (28.57%). Marker-trait association (MTA) and Single Marker Analysis (SMA) revealed that the SSR marker pPGPseq5D05 is significantly associated with both LLS (15.8% PVE) and rust (17.5% PVE) resistance, whereas, the marker IPAHM103 is tightly linked with rust resistance (26.8% PVE) alone. In silico analysis revealed that the marker gene for IPAHM103 is a zinc finger protein and the marker gene for pPGPseq5D05 is an ADP-ribosylation factor GTPase-activating protein. Both these protein products impart resistance or tolerance to biotic stress in crop plants. Two other markers namely, GMLQ975 and pPGPseq13A10 were also found to be associated with LLS resistance explaining MTA up to 60%., Conclusion: These gene specific markers will enable us to screen more number of germplasm lines or newly developed lines in MAS schemes for LLS and rust resistance using a wide range of resistant sources., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Pathogen perception generates the activation of signal transduction cascades to host defense. White pine blister rust (WPBR) is caused by Cronartium ribicola J.C. Fisch and affects a number of species of Pinus . One of the most severely affected species is Pinus albicaulis Engelm (whitebark pine). WPBR resistance in the species is a polygenic and complex trait that requires an optimized immune response. We identified early responses in 2-year-old seedlings after four days of fungal inoculation and compared the underlying transcriptomic response with that of healthy non-inoculated individuals. A de novo transcriptome assembly was constructed with 56,796 high quality-annotations derived from the needles of susceptible and resistant individuals in a resistant half-sib family. Differential expression analysis identified 599 differentially expressed transcripts, from which 375 were upregulated and 224 were downregulated in the inoculated seedlings. These included components of the initial phase of active responses to abiotic factors and stress regulators, such as those involved in the first steps of flavonoid biosynthesis. Four days after the inoculation, infected individuals showed an overexpression of chitinases, reactive oxygen species (ROS) regulation signaling, and flavonoid intermediates. Our research sheds light on the first stage of infection and emergence of disease symptoms among whitebark pine seedlings. RNA sequencing (RNA-seq) data encoding hypersensitive response, cell wall modification, oxidative regulation signaling, programmed cell death, and plant innate immunity were differentially expressed during the defense response against C. ribicola .

Leaf rust caused by the pathogen Puccinia triticina ( Pt ) is a destructive fungal disease of wheat that occurs in almost all wheat-growing areas across the globe. Genetic resistance has proven to be the best solution to mitigate the disease. Wheat breeders are continuously seeking new diversified and durable sources of resistance to use in developing new varieties. We developed recombinant inbred line (RIL) populations from two leaf rust-resistant genotypes (Kenya Kudu and AUS12568) introduced from Kenya to identify and characterize resistance to Pt and to develop markers linked closely to the resistance that was found. Our studies detected four QTL conferring adult plant resistance (APR) to leaf rust. Two of these loci are associated with known genes, Lr46 and Lr68 , residing on chromosomes 1B and 7B, respectively. The remaining two, QLrKK_2B and QLrAus12568_5A , contributed by Kenya Kudu and AUS12568 respectively, are putatively new loci for Pt resistance. Both QLrKK_2B and QLrAus12568_5A were found to interact additively with Lr46 in significantly reducing the disease severity at adult plant growth stages in the field. We further developed a suite of six closely linked markers within the QLrAus12568_5A locus and four within the QLrKK_2B region. Among these, markers sunKASP_522 and sunKASP_524 , flanking QLrAus12568_5A , and sunKASP_536 , distal to QLrKK_2B , were identified as the most closely linked and reliable for marker-assisted selection. The markers were validated on a selection of 64 Australian wheat varieties and found to be polymorphic and robust, allowing for clear allelic discrimination. The identified new loci and linked molecular markers will enable rapid adoption by breeders in developing wheat varieties carrying diversified and durable resistance to leaf rust.

Key Message: An adult plant gene for resistance to stripe rust was narrowed down to the proximal one-third of the 2N v S segment translocated from Aegilops ventricosa to wheat chromosome arm 2AS, and based on the gene expression analysis, two candidate genes were identified showing a stronger response at the adult plant stage compared to the seedling stage. The 2N v S translocation from Aegilops ventricosa, known for its resistance to various diseases, has been pivotal in global wheat breeding for more than three decades. Here, we identified an adult plant resistance (APR) gene in the 2N v S segment in wheat line K13-868. Through fine mapping in a segregating near-isogenic line (NIL) derived population of 6389 plants, the candidate region for the APR gene was narrowed down to between 19.36 Mb and 33 Mb in the Jagger reference genome. Transcriptome analysis in NILs strongly suggested that this APR gene conferred resistance to stripe rust by triggering plant innate immune responses. Based on the gene expression analysis, two disease resistance-associated genes within the candidate region, TraesJAG2A03G00588940 and TraesJAG2A03G00590140, exhibited a stronger response to Puccinia striiformis f. sp. tritici (Pst) infection at the adult plant stage than at the seedling stage, indicating that they could be potential candidates for the resistance gene. Additionally, we developed a co-dominant InDel marker, InDel_31.05, for detecting this APR gene. Applying this marker showed that over one-half of the wheat varieties approved in 2021 and 2022 in Sichuan province, China, carry this gene. Agronomic trait evaluation of NILs indicated that the 2N v S segment effectively mitigated the negative effects of stripe rust on yield without affecting other important agronomic traits. This study provided valuable insights for cloning and breeding through the utilization of the APR gene present in the 2N v S segment., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To better understand how the pathogenicity of the oat crown rust pathogen Puccinia coronata f. sp. avenae ( Pca ) has changed in the United States, 30 years of United States Department of Agriculture (USDA) survey isolates ( n = 5,456) tested on 30 to 40 differential lines were analyzed for overall and Pc -resistance-gene-specific virulence trends and correlations. Pca is incredibly pathologically diverse, with 88% of races represented by a single isolate. There are a slightly higher proportion of unique races from the Northern region of the United States, and for one fourth of the years, Northern region isolates were significantly more virulent than Southern isolates, which supports the idea that sexual recombination in this region is mediated by the alternate host as a major factor in creating new races. However, there is also support for regular isolate movement between North and South regions as isolates in the United States are steadily accumulating virulences at a rate of 0.35 virulences per year. Virulence significantly increased for 23 and decreased for four of the 40 differential lines. In the past few years, virulence has reached 90% or greater for 16 differential lines. There were also strong correlations in virulence for certain Pc genes that are likely identical, allelic, or target the same or closely linked pathogen effectors (e.g., Pc39 , Pc55 , and Pc71 ), and the results were largely in concordance with recent genome-wide association study (GWAS) effector studies using USDA isolate subsets. Understanding changes in Pca pathogenicity is essential for the responsible deployment and management of Pc resistance genes for sustainable and profitable oat production., Competing Interests: The author(s) declare no conflict of interest.

Coffea spp. is the source of one of the most widely consumed beverages in the world. However, the cultivation of this crop is threatened by Hemileia vastatrix Berk & Broome, a fungal disease, which reduces the productivity and can cause significant economic losses. In this protocol, coffee leaf segment derived from a chemical mutagenesis process are inoculated with uredospores of the pathogen. Subsequently, the gene expression changes are analyzed over the time (0, 5, 24, 48, and 120 h) using quantitative real-time polymerase chain reaction (RT-qPCR). The procedures and example data are presented for expression analysis in the CaWRKY1 gene. This procedure can be applied for quantitative analysis of other genes of interest to coffee breeders and scientists for elucidating the molecular mechanisms involved in the interaction between the plant and pathogen, potentially leading to the development of more efficient approaches for managing this disease., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

All native North American white pines are highly susceptible to white pine blister rust (WPBR) caused by Cronartium ribicola . Understanding genomic diversity and molecular mechanisms underlying genetic resistance to WPBR remains one of the great challenges in improvement of white pines. To compare major gene resistance (MGR) present in two species, southwestern white pine ( Pinus strobiformis ) Cr3 and limber pine ( P . flexilis ) Cr4 , we performed association analyses of Cr3 -controlled resistant traits using single nucleotide polymorphism (SNP) assays designed with Cr4 -linked polymorphic genes. We found that ∼70% of P. flexilis SNPs were transferable to P. strobiformis . Furthermore, several Cr4 -linked SNPs were significantly associated with the Cr3 -controlled traits in P. strobiformis families. The most significantly associated SNP (M326511_1126R) almost colocalized with Cr4 on the Pinus consensus linkage group 8, suggesting that Cr3 and Cr4 might be the same R locus, or have localizations very close to each other in the syntenic region of the P. strobiformis and P. flexilis genomes. M326511_1126R was identified as a nonsynonymous SNP, causing amino acid change (Val 376 Ile) in a putative pectin acetylesterase, with coding sequences identical between the two species. Moreover, top Cr3 -associated SNPs were further developed as TaqMan genotyping assays, suggesting their usefulness as marker-assisted selection (MAS) tools to distinguish genotypes between quantitative resistance and MGR. This work demonstrates the successful transferability of SNP markers between two closely related white pine species in the hybrid zone, and the possibility for deployment of MAS tools to facilitate long-term WPBR management in P. strobiformis breeding and conservation.

Critical transitions, sudden responses to slow changes in environmental drivers, are inherent in many dynamic processes, prompting a search for early warning signals. We apply this framework to understanding the coffee rust disease, which experienced an unprecedented outbreak in Mesoamerica in 2012-2013, likely a critical transition. Based on monthly infection data from 128 study quadrats in a 45-ha plot in southern Mexico from 2014 to 2020, we find that the persistent seasonal epidemic following the initial outbreak collapses in an evident subsequent critical transition. Characteristic signals of "critical slowing down" precede this collapse and are correlated with reduced rainfall, as expected from climate change, and planting of rust-resistant varieties, an ongoing management intervention. Recoveries from catastrophes may themselves be experienced as a critical transition and managers should consider the larger dynamical landscape for the possibility of subsequent transitions. Early warning signals could therefore be useful when evaluating mitigation effectiveness., (© 2022. The Author(s).)

Pathogenesis-related 1 (PR-1) proteins, which are defense proteins in plant-pathogen interactions, play an important role in the resistance and defense of plants against diseases. Blister blight disease is caused by Exobasidium vexans Massee and a major leaf disease of tea plants ( Camellia sinensis (L.) O. Kuntze). However, the systematic characterization and analysis of the PR-1 gene family in tea plants is still lacking, and the defense mechanism of this family remains unknown. In this study, 17 CsPR-1 genes were identified from the tea plant genome and classified into five groups based on their signal peptide, isoelectric point, and C-terminus extension. Most of the CsPR-1 proteins contained an N-terminal signal peptide and a conserved PR-1 like domain. CsPR-1 genes comprised multiple cis -acting elements and were closely related to the signal-transduction pathways involving TCA, NPR1, EDS16, BGL2, PR4, and HCHIB. These characteristics imply an important role of the genes in the defense of the tea plant. In addition, the RNA-seq data and real-time PCR analysis demonstrated that the CsPR-1-2 , -4, -6 , -7 , -8 , -9 , -10 , -14 , -15, and -17 genes were significantly upregulated under tea blister-blight stress. This study could help to increase understanding of CsPR-1 genes and their defense mechanism in response to tea blister blight.

A population of 239 perennial ryegrass ( Lolium perenne L.) genotypes was analyzed to identify marker-trait associations for crown rust ( Puccinia coronata f. sp. lolii ) and brown rust ( Puccinia graminis f. sp. loliina ) resistance. Phenotypic data from field trials showed a low correlation ( r = 0.17) between the two traits. Genotypes were resequenced, and a total of 14,538,978 SNPs were used to analyze population structure, linkage disequilibrium (LD), and for genome-wide association study. The SNP heritability ( h 2 SNP ) was 0.4 and 0.8 for crown and brown rust resistance, respectively. The high-density SNP dataset allowed us to estimate LD decay with the highest possible precision to date for perennial ryegrass. Results showed a low LD extension with a rapid decay of r 2 value below 0.2 after 520 bp on average. Additionally, QTL regions for both traits were detected, as well as candidate genes by applying Genome Complex Trait Analysis and Multi-marker Analysis of GenoMic Annotation. Moreover, two significant genes, LpPc6 and LpPl6 , were identified for crown and brown rust resistance, respectively, when SNPs were aggregated to the gene level. The two candidate genes encode proteins with phosphatase activity, which putatively can be induced by the host to perceive, amplify and transfer signals to downstream components, thus activating a plant defense response.

Fungal infections are responsible for high morbidity and mortality in neonatal patients, especially in premature newborns. Infections in neonates caused by Cryptococcus spp. are rare, but it has occurred in an immunocompromised population. This study aims to describe the isolation of Cryptococcus liquefaciens from the hands of a health professional in a neonatal intensive care unit, and to evaluate the production of biofilm and virulence factors and susceptibility to antifungals. Antifungal susceptibility tests were performed according to Clinical and Laboratory Standard Institute document M27-A3. Thermotolerance virulence factors and DNase, phospholipase, proteinase, and hemolytic activities were verified through phenotypic tests; biofilm was evaluated by determining the metabolic activity and biomass. The isolate did not produce any of the tested enzymes and was susceptible to all antifungals (amphotericin B, fluconazole, and micafungin). The growth at 37 °C was very weak; however, the isolate showed a strong biomass production and low metabolic activity. This is the first report of C. liquefaciens isolated from the hands of a health professional. The isolate did not express any of the studied virulence factors in vitro, except for the low growth at 37 °C in the first 48 h, and the strong production of biofilm biomass. Cryptococcus liquefaciens can remain in the environment for a long time and is a human pathogen because it tolerates temperature variations. This report draws attention to the circulation of rare species in critical locations, information that may help in a fast and correct diagnosis and, consequently, implementation of an appropriate treatment., (© 2021. Sociedade Brasileira de Microbiologia.)

Background: Sporisorium scitamineum is the causative agent of smut disease in sugarcane. The tricky life cycle of S. scitamineum consists of three distinct growth stages: diploid teliospores, haploid sporidia and dikaryotic mycelia. Compatible haploid sporidia representing opposite mating types (MAT-1 and MAT-2) of the fungus fuse to form infective dikaryotic mycelia in the host tissues, leading to the development of a characteristic whip shaped sorus. In this study, the transition of distinct stages of in vitro life cycle and in planta developmental stages of S. scitamineum are presented by generating stable GFP transformants of S. scitamineum., Methods and Results: Haploid sporidia were isolated from the teliospores of Ss97009, and the opposite mating types (MAT-1 and MAT-2) were identified by random mating assay and mating type-specific PCR. Both haploid sporidia were individually transformed with pNIIST plasmid, harboring an enhanced green fluorescent protein (eGFP) gene and hygromycin gene by a modified protoplast-based PEG-mediated transformation method. Thereafter, the distinct in vitro developmental stages including fusion of haploid sporidia and formation of dikaryotic mycelia expressing GFP were demonstrated. To visualize in planta colonization, transformed haploids (MAT-1gfp and MAT-2gfp) were fused and inoculated onto the smut susceptible sugarcane cultivar, Co 97009 and examined microscopically at different stages of colonization. GFP fluorescence-based analysis presented an extensive fungal colonization of the bud surface as well as inter- and intracellular colonization of the transformed S. scitamineum in sugarcane tissues during initial stages of disease development. Noticeably, the GFP-tagged S. scitamineum led to the emergence of smut whips, which established their pathogenicity, and demonstrated initial colonization, active sporogenesis and teliospore maturation stages., Conclusion: Overall, for the first time, an efficient protoplast-based transformation method was employed to depict clear-cut developmental stages in vitro and in planta using GFP-tagged strains for better understanding of S. scitamineum life cycle development., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae , a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

The biotrophic fungal pathogen Ustilago maydis causes common smut in maize, forming tumors on all aerial organs, especially on reproductive organs, leading to significant reduction in yield and quality defects. Resistance to U. maydis is thought to be a quantitative trait, likely controlled by many minor gene effects. However, the genes and the underlying complex mechanisms for maize resistance to U. maydis remain largely uncharacterized. Here, we conducted comparative transcriptome and metabolome study using a pair of maize lines with contrast resistance to U. maydis post-infection. WGCNA of transcriptome profiling reveals that defense response, photosynthesis, and cell cycle are critical processes in maize response to U. maydis , and metabolism regulation of glycolysis, amino acids, phenylpropanoid, and reactive oxygen species are closely correlated with defense response. Metabolomic analysis supported that phenylpropanoid and flavonoid biosynthesis was induced upon U. maydis infection, and an obviously higher content of shikimic acid, a key compound in glycolysis and aromatic amino acids biosynthesis pathways, was detected in resistant samples. Thus, we propose that complex gene co-expression and metabolism networks related to amino acids and ROS metabolism might contribute to the resistance to corn smut.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta , which may conduct fungus growth and infection at the initial stage of stem-gall formation.

Plant associated microbiomes are known to confer fitness advantages to the host. Understanding how plant factors including biochemical traits influence host associated microbiome assembly could facilitate the development of microbiome-mediated solutions for sustainable plant production. Here, we examined microbial community structures of a set of well-characterized Arabidopsis thaliana mutants disrupted in metabolic pathways for the production of glucosinolates, flavonoids, or a number of defense signalling molecules. A. thaliana lines were grown in a natural soil and maintained under greenhouse conditions for 4 weeks before collection of roots for bacterial and fungal community profiling. We found distinct relative abundances and diversities of bacterial and fungal communities assembled in the individual A. thaliana mutants compared to their parental lines. Bacterial and fungal genera were mostly enriched than depleted in secondary metabolite and defense signaling mutants, except for flavonoid mutations on fungi communities. Bacterial genera Azospirillum and Flavobacterium were significantly enriched in most of the glucosinolate, flavonoid and signalling mutants while the fungal taxa Sporobolomyces and Emericellopsis were enriched in several glucosinolates and signalling mutants. Whilst the present study revealed marked differences in microbiomes of Arabidopsis mutants and their parental lines, it is suggestive that unknown enzymatic and pleiotropic activities of the mutated genes could contribute to the identified host-associated microbiomes. Notwithstanding, this study revealed interesting gene-microbiota links, and thus represents valuable resource data for selecting candidate A. thaliana mutants for analyzing the links between host genetics and the associated microbiome., Competing Interests: The authors declare that they have no conflict of interest.

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection., (© 2021. The Author(s).)

Triticale (× Triticosecale Wittmack) is a commercial hybrid harboring wheat (Triticum sp.) and rye (Secale cereale L.) genomes. The limited genetic diversity of this crop resulted in the collapse of fungal disease resistance. Leaf rust disease, caused by Puccinia triticina Eriks., is reported to reduce the triticale yield significantly (more than 30%). There is a need to enlarge the genetic variability of this crop including leaf resistance genes. The main aim of this research was to evaluate the leaf rust resistance of the offspring of translocation lines of triticale carrying chromatin of Ae. tauschii and Ae. kotschyi. A reaction of seedlings of 200 plants of two triticale-Aegilops translocation lines (Bogo-2D t .2R and Sekundo-2S k .2R) was compared after inoculation with a natural mixture of P. triticina races, specific to triticale in controlled condition. Before inoculation, each plant was screened using molecular cytogenetics and molecular markers linked to leaf rust resistance genes. The presence of Aegilops chromosome segments was confirmed using genomic in situ hybridization (GISH). Lr39 and Lr54 leaf rust resistance genes were identified using Xgdm35 and S14 molecular markers, respectively. After inoculation, a significant improvement of resistance severity was observed in Sekundo-2S k .2R in comparison with triticale cv. Sekundo plants. The resistance level of Bogo-2D t .2R did not differ compared with triticale cv. Bogo plants. It was shown that Lr39 gene did not increase the leaf rust resistance level of triticale cv. Bogo., (© 2021. The Author(s).)

TaLHY is an MYB transcription factor (TF) that is upregulated by salicylic acid induction and shows circadian rhythms. However, the study of the upstream regulatory factors is still unclear. In this study, we cloned the promoter sequence of the TaLHY homologous genes, verified the activity of the promoters, and identified important regions that affect promoter activity. Furthermore, we explored a possible upstream regulator of TaLHY, named TaWRKY10, which played a key role in the expression of TaLHY. We found that the three promoters pTaLHYa, pTaLHYb, and pTaLHYd had transcriptional activity in wheat protoplasts. All three promoters have W-Box, which can bind to WRKY TFs. Using virus-induced gene silencing (VIGS), after silencing TaWRKY10, the resistance of ChuanNong 19 (CN19) to stripe rust pathogen strain CYR32 was lost, and the expression level of the TaLHY homologous gene decreased. At the same time, in wheat protoplasts, the transcriptional activity of TaLHY homologous promoters improved after TaWRKY10 overexpression. This indicates that TaWRKY10 is a key gene for wheat immune response to stripe rust, and this gene may bind to TaLHYa, TaLHYb, and TaLHYd promoters to regulate the expression of TaLHY., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Dendrobium catenatum belongs to the Orchidaceae, and is a precious Chinese herbal medicine. In the past 20 years, D. catenatum industry has developed from an endangered medicinal plant to multi-billion dollar grade industry. The necrotrophic pathogen Sclerotium delphinii has a devastating effection on over 500 plant species, especially resulting in widespread infection and severe yield loss in the process of large-scale cultivation of D. catenatum. It has been widely reported that Jasmonate (JA) is involved in plant immunity to pathogens, but the mechanisms of JA-induced plant resistance to S. delphinii are unclear., Results: In the present study, the role of JA in enhancing D. catenatum resistance to S. delphinii was investigated. We identified 2 COI1, 13 JAZ, and 12 MYC proteins in D. catenatum genome. Subsequently, systematic analyses containing phylogenetic relationship, gene structure, protein domain, and motif architecture of core JA pathway proteins were conducted in D. catenatum and the newly characterized homologs from its closely related orchid species Phalaenopsis equestris and Apostasia shenzhenica, along with the well-investigated homologs from Arabidopsis thaliana and Oryza sativa. Public RNA-seq data were investigated to analyze the expression patterns of D. catenatum core JA pathway genes in various tissues and organs. Transcriptome analysis of MeJA and S. delphinii treatment showed exogenous MeJA changed most of the expression of the above genes, and several key members, including DcJAZ1/2/5 and DcMYC2b, are involved in enhancing defense ability to S. delphinii in D. catenatum., Conclusions: The findings indicate exogenous MeJA treatment affects the expression level of DcJAZ1/2/5 and DcMYC2b, thereby enhancing D. catenatum resistance to S. delphinii. This research would be helpful for future functional identification of core JA pathway genes involved in breeding for disease resistance in D. catenatum., (© 2021. The Author(s).)

Key Message: Individual stem rust resistance genes could be directly mapped within self-incompatible rye populations. Genetic resources of rye (Secale cereale L.) are cross-pollinating populations that can be highly diverse and are naturally segregating. In this study, we show that this segregation could be used for mapping stem rust resistance. Populations of pre-selected donors from the Russian Federation, the USA and Austria were tested on a single-plant basis for stem rust resistance by a leaf-segment test with three rust isolates. Seventy-four plants per population were genotyped with a 10 K-SNP chip. Using cumulative logit models, significant associations between the ordinal infection score and the marker alleles could be found. Three different loci (Pgs1, Pgs2, Pgs3) in three populations were highly significant, and resistance-linked markers could be validated with field experiments of an independent seed sample from the original population and were used to fix two populations for resistance. We showed that it is possible to map monogenically inherited seedling resistance genes directly in genetic resources, thus providing a competitive alternative to linkage mapping approaches that require a tedious and time-consuming inbreeding over several generations.

Key Message: A large genomic region spanning over 300 Mb on chromosome 6A under intense artificial selection harbors multiple loci associated with favorable traits including stripe rust resistance in wheat. The development of resistance cultivars can be an optimal strategy for controlling wheat stripe rust disease. Although loci for stripe rust resistance have been identified on chromosome 6A in previous studies, it is unclear whether these loci span a common genetic interval, and few studies have attempted to analyze the haplotype changes that have accompanied wheat improvement over the period of modern breeding. In this study, we used F 2:3 families and F 6:7 recombinant inbred lines (RILs) derived from a cross between a resistant CIMMYT wheat accession P10090 and the susceptible landrace Mingxian 169 to improve the resolution of the QTL on chromosome 6A. The co-located QTL, designated as YrP10090, was flanked by SNP markers AX-94460938 and AX-110585473 with a genetic interval of 3.5 cM, however, corresponding to a large physical distance of over 300 Mb in RefSeq v.1.0 (positions 107.1-446.5 Mb). More than 1,300 SNP markers in this genetic region were extracted for haplotype analysis in a panel of 1,461 worldwide common wheat accessions, and three major haplotypes (Hap1, Hap2, and Hap3) were identified. The favorable haplotype Hap1 associated with stripe rust resistance exhibited a large degree of linkage disequilibrium. Selective sweep analyses were performed between different haplotype groups, revealing specific genomic regions with strong artificial selection signals. These regions harbored multiple desirable traits associated with resilience to environmental stress, different yield components, and quality characteristics. P10090 and its derivatives that carry the desirable haplotype can provide a concrete foundation for bread wheat improvement including the genomic selection.

Key Message: Discovery of two rust resistance genes, R 17 and R 18 , from the sunflower lines introduced from South Africa and genetic mapping of them to sunflower chromosome 13. Rust, caused by the fungus Puccinia helianthi Schw., is one of the most serious diseases of sunflower in the world. The rapid changes that occur in the virulence characteristics of pathogen populations present a continuous threat to the effectiveness of existing rust-resistant hybrids. Thus, there is a continued need for the characterization of genetically diverse sources of rust resistance. In this study, we report to identify two new rust resistance genes, R 17 and R 18 , from the sunflower lines, KP193 and KP199, introduced from South Africa. The inheritance of rust resistance was investigated in both lines using two mapping populations developed by crossing the resistant plants selected from KP193 and KP199 with a common susceptible parent HA 89. The F 2 populations were first genotyped using genotyping by sequencing for mapping of the rust genes and further saturated with markers in the target region. Molecular mapping positioned the two genes at the lower end of sunflower chromosome 13 within a large gene cluster. Two co-segregating SNP markers, SFW01497 and SFW08875, were distal to R 17 at a 1.9 cM genetic distance, and a cluster of five co-segregating SNPs was proximal to R 17 at 0.7 cM. R 18 co-segregated with the SNP marker SFW04317 and was proximal to two cosegregating SNPs, SFW01497 and SFW05453, at 1.9 cM. These maps provide markers for stacking R 17 or R 18 with other broadly effective rust resistance genes to extend the durability of rust resistance. The relationship of the six rust resistance genes in the cluster was discussed.

Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here, we identified multiple novel members in the western white pine ( Pinus monticola ) PmPR10 family by bioinformatic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of 10 PmPR10 genes, PmPR10-3.1 was selected and expressed in Escherichia coli. The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens Cronartium ribicola , Phoma exigua , and Phoma argillacea by in-vitro anti-fungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within PmPR10-3.1 in a collection of P. monticola seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying P. monticola QDR and imply that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.

Key Message: An adult plant stripe rust resistance gene Yr75 was located on the long arm of chromosome 7A. Fine mapping of the region identified markers closely linked with Yr75. Australian wheat cultivar Axe produced resistant to moderately resistant stripe rust responses under field conditions and was exhibiting seedling responses varying from 33C to 3+ under greenhouse conditions. Experiments covering tests at different growth stages (2nd, 3rd and 4th leaf stages) demonstrated the clear expression of resistance at the 4th leaf stage under controlled-environment greenhouse conditions. A recombinant inbred line (RIL) population was developed from the Axe/Nyabing-3 (Nyb) cross. Genetic analysis of Axe/Nyb RIL population in the greenhouse at the 4th leaf stage showed monogenic inheritance of stripe rust resistance. Selective genotyping using the iSelect 90 K Infinium SNP genotyping array was performed, and the resistance locus was mapped to the long arm of chromosome 7A and named Yr75. The Axe/Nyb RIL population was genotyped using a targeted genotype-by-sequencing assay, and the resistance-linked SNPs were converted into kompetitive allele-specific PCR (KASP) markers. These markers were tested on the entire Axe/Nyb RIL population, and markers sunKASP_430 and sunKASP_427 showed close association with Yr75 in the Axe/Nyb RIL population. A high-resolution mapping family of 1032 F 2 plants from the Axe/Nyb cross was developed and genotyped with sunKASP_430 and sunKASP_427, and these markers flanked Yr75 at 0.3 cM and 0.4 cM, respectively. These markers cover 1.24 Mb of the physical map of Chinese Spring, and this information will be useful for map-based cloning of Yr75.

Key message The stripe rust resistance gene Yr34 was transferred to polyploid wheat chromosome 5AL from T. monococcum and has been used for over two centuries.Wheat stripe (or yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is currently among the most damaging fungal diseases of wheat worldwide. In this study, we report that the stripe rust resistance gene Yr34 (synonym Yr48) is located within a distal segment of the cultivated Triticum monococcum subsp. monococcum chromosome 5A m L translocated to chromosome 5AL in polyploid wheat. The diploid wheat species Triticum monococcum (genome A m A m ) is closely related to T. urartu (donor of the A genome to polyploid wheat) and has good levels of resistance against the stripe rust pathogen. When present in hexaploid wheat, the T. monococcum Yr34 resistance gene confers a moderate level of resistance against virulent Pst races present in California and the virulent Chinese race CYR34. In a survey of 1,442 common wheat genotypes, we identified 5A m L translocations of fourteen different lengths in 17.5% of the accessions, with higher frequencies in Europe than in other continents. The old European wheat variety "Mediterranean" was identified as a putative source of this translocation, suggesting that Yr34 has been used for over 200 years. Finally, we designed diagnostic CAPS and sequenced-based markers that will be useful to accelerate the deployment of Yr34 in wheat breeding programs to improve resistance to this devastating pathogen.

Biotrophic plant pathogens secrete effector proteins to manipulate the host physiology. Effectors suppress defenses and induce an environment favorable to disease development. Sequence-based prediction of effector function is impeded by their rapid evolution rate. In the maize pathogen Ustilago maydis, effector-coding genes frequently organize in clusters. Here we describe the functional characterization of the pleiades, a cluster of ten effector genes, by analyzing the micro- and macroscopic phenotype of the cluster deletion and expressing these proteins in planta. Deletion of the pleiades leads to strongly impaired virulence and accumulation of reactive oxygen species (ROS) in infected tissue. Eight of the Pleiades suppress the production of ROS upon perception of pathogen associated molecular patterns (PAMPs). Although functionally redundant, the Pleiades target different host components. The paralogs Taygeta1 and Merope1 suppress ROS production in either the cytoplasm or nucleus, respectively. Merope1 targets and promotes the auto-ubiquitination activity of RFI2, a conserved family of E3 ligases that regulates the production of PAMP-triggered ROS burst in plants., Competing Interests: The authors have declared that no competing interests exist.

Plant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target.

Organisms are constantly challenged by pathogens and pests, which can drive the evolution of growth-defense strategies. Plant stomata are essential for gas exchange during photosynthesis and conceptually lie at the intersection of the physiological demands of growth and exposure to foliar fungal pathogens. Generations of natural selection for locally adapted growth-defense strategies can eliminate variation between traits, potentially masking trade-offs and selection conflicts that may have existed in the past. Hybrid populations offer a unique opportunity to reset the clock on selection and to study potentially maladaptive trait variation before selection removes it. We study the interactions of growth, stomatal, ecopysiological, and disease resistance traits in poplars (Populus) after infection by the leaf rust Melampsora medusae. Phenotypes were measured in a common garden and genotyped at 227K SNPs. We isolate the effects of hybridization on trait variance, discover correlations between stomatal, ecophysiology, and disease resistance, examine trade-offs and selection conflicts, and explore the evolution of growth-defense strategies potentially mediated by selection for stomatal traits on the upper leaf surface. These results suggest an important role for stomata in determining growth-defense strategies in organisms susceptible to foliar pathogens, and reinforces the contribution of hybridization studies toward our understanding of trait evolution., (© 2021 The Authors. Evolution © 2021 The Society for the Study of Evolution.)