A NAC Transcription Factor TuNAC69 Contributes to ANK-NLR-WRKY NLR-Mediated Stripe Rust Resistance in the Diploid Wheat Triticum urartu

Stripe rust is one of the most devastating diseases in wheat. Nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain receptors (NLRs) recognize pathogenic effectors and trigger plant immunity. We previously identified a unique NLR protein YrU1 in the diploid wheat Triticum urartu, which contains an N-terminal ANK domain and a C-terminal WRKY domain and confers disease resistance to stripe rust fungus Puccinia striiformis f. sp. Tritici (Pst). However, how YrU1 functions in disease resistance is not clear. In this study, through the RNA-seq analysis, we found that the expression of a NAC member TuNAC69 was significantly up-regulated after inoculation with Pst in the presence of YrU1. TuNAC69 was mainly localized in the nucleus and showed transcriptional activation in yeast. Knockdown TuNAC69 in diploid wheat Triticum urartu PI428309 that contains YrU1 by virus-induced gene silencing reduced the resistance to stripe rust. In addition, overexpression of TuNAC69 in Arabidopsis enhanced the resistance to powdery mildew Golovinomyces cichoracearum. In summary, our study indicates that TuNAC69 participates in the immune response mediated by NLR protein YrU1, and likely plays an important role in disease resistance to other pathogens.

Virulence phenotypes of the wheat leaf rust pathogen, Puccinia triticina, in the United States 2018-2020

Collections of wheat leaves infected with the leaf rust fungus, Puccinia triticina, were obtained from the southeastern states, the Ohio Valley, the Great Plains, and Washington in 2018, 2019 and 2020 to determine the prevalent virulence phenotypes in the wheat growing regions of the United States. In the hard red winter wheat region of the southern and mid Great Plains, MNPSD, and MPPSD were the two most common phenotypes in 2018 and 2019. In 2020 BBBQD with high virulence to durum wheat was the most common phenotype in the southern Great Plains. In the hard red spring wheat region of the northern Great Plains, MNPSD, MPPSD, MBDSD, and TBBGS were the predominant phenotypes. In the soft red winter wheat region of the southeastern states and Ohio Valley region, MBTNB, MCTNB, and MNPSD were the three most common phenotypes. Collections in Washington had phenotypes LBDSG, LCDSG, LCDJG, and MBDSB that were not found in any other region. Isolates with virulence to Lr11 were most frequent in the southeastern states, and Ohio Valley regions. The frequency of isolates with virulence to Lr39 was highest in the Great Plains region and frequency of isolates with virulence to Lr21 was highest in the northern Great Plains region. Selection of virulence phenotypes by leaf rust resistance genes in the different market classes of wheat, combined with the effects of clonal reproduction, overwintering in southern regions, and low migration between the Great Plains region and eastern wheat producing regions, has maintained the different P. triticina populations in the United States.

Transcriptome Analysis of Eucalyptus grandis Implicates Brassinosteroid Signaling in Defense Against Myrtle Rust (Austropuccinia psidii)

Eucalyptus grandis, in its native Australian range, varies in resistance to Austropuccinia psidii (syn. Puccinia psidii). The biotrophic rust fungus, A. psidii is the causal agent of myrtle rust and poses a serious threat to Australian biodiversity. The pathogen produces yellow pustules of urediniospores on young leaves and shoots, resulting in shoot tip dieback, stunted growth, and death. Dissecting the underlying mechanisms of resistance against this pathogen will contribute to improved breeding and control strategies to mitigate its devastating effects. The aim of this study was to determine the molecular dialogue between E. grandis and A. psidii, using an RNA-sequencing approach. Resistant and susceptible E. grandis seedlings grown from seed collected across its natural range were inoculated with the pandemic biotype of A. psidii. The leaf tissue was harvested at 12-h post inoculation (hpi), 1-day post inoculation (dpi), 2-dpi and 5-dpi and subjected to RNA-sequencing using Illumina 50 bp PE reads to a depth of 40 million reads per sample. Differential gene expression and gene ontology enrichment indicated that the resistant seedlings showed controlled, coordinated responses with a hypersensitive response, while the susceptible seedlings showed no systemic response against myrtle rust. Brassinosteroid signaling was apparent as an enriched term in the resistant interaction at 2-dpi, suggesting an important role of this phytohormone in defense against the pathogen. Brassinosteroid mediated signaling genes were also among the candidate genes within two major disease resistance loci (Puccinia psidii resistance), Ppr3 and Ppr5. While brassinosteroids have been tagged as positive regulators in other plant disease resistance interactions, this is the first report in the Eucalyptus – Austropuccinia psidii interaction. Furthermore, several putative resistance genes, underlying known resistance loci and implicated in the interaction have been identified and highlighted for future functional studies. This study provided further insights into the molecular interactions between E. grandis and A. psidii, contributing to our understanding of this pathosystem.

First Report of Puccinia thaliae Causing leaf rust on Canna indica in Malaysia

Canna indica L. (family Cannaceae), locally known as Bunga Kana, is a perennial plant grown as a source of starch and for ornamental purposes in Malaysia. During June 2021, Bunga Kana with rust symptoms and signs were collected from the Universiti Malaysia Sabah in the province of Sabah. The severity was 95%, and the incidence was 90%. Yellow uredinia were observed primarily on the abaxial surface of the leaves. As the disease progressed, leaves were covered with coalescing pustules, and chlorosis and brown necrosis developed. Microscopic examination of pustules revealed the presence of urediniospores and teliospores. Urediniospores were round to ovoid in shape, yellow, and echinulate, 17.7 to 24.6 x 26.8 to 45.2 μm, with two equatorial pores. Teliospores were elongate-clavate, with rounded apex, yellow contents, 18.3 x 20.2 to 45.8 x 53.9 μm, with a short pedicel. Yellow urediniospores were collected using a fine brush, and genomic DNA was extracted using lysis buffer [Tris-HCl (0.1M, pH 9.5), NaCl (1M), EDTA (0.5M, pH 8)] prior to heating at 95°C for 10 min. KOD One PCR master mix containing hot-start modified KOD DNA polymerase was used for PCR amplification. The 28S ribosomal RNA gene was amplified using primers Rust28SF (Aime et al. 2018) and LR5 (Vilgalys and Hester 1990). BLASTn analysis of the newly generated 28S ribosomal RNA gene (OK462969) in GenBank revealed a 99% sequence identity to Puccinia thaliae Dietel (JX206994 of 28S ribosomal RNA gene). The morphological and molecular characterization of the rust fungus matched P. thaliae described by Padamsee and McKenzie (2012). Koch's postulates were performed with spray inoculations of urediniospores suspended in water (106 spores/ml) on leaves of three healthy Bunga Kana plants, while water was sprayed on three additional Bunga Kana plants which served as control. The inoculated Bunga Kana plants were covered with plastics for 48 h at 25°C in the dark, and then placed in the greenhouse. Symptoms and signs similar to those of the field collection occurred after 13 days post inoculation. No symptoms occurred on controls. Leaf rust on Bunga Kana plants caused by P. thaliae has been reported in Europe (Talhinhas et al. 2016), Hawaii (Nelson 2013), India (Gopi et al. 2014), Mexico (Cedas de Jesús et al. 2018), Nepal (Adhikari and Durrieu 2016), New Zealand (Padamsee and McKenzie 2012), Singapore (Neo and Tham 2010) and South Africa (van Jaarsveld et al. 2006) over the past fifteen years. To our knowledge, this is the first report of P. thaliae causing leaf rust on C. indica in Malaysia. Our findings expand the geographic range of P. thaliae and indicate it could be a potential threat limiting the starch production of C. indica in Malaysia.

Puccinia tuberosa, a new species of rust fungus on Allium tuberosum in Asia

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The Barley HvSTP13GR mutant triggers resistance against biotrophic fungi

High-yielding and stress resistant crops are essential to ensure future food supply. Barley is an important crop to feed livestock and to produce malt, but the annual yield is threatened by pathogen infections. Pathogens can trigger an altered sugar partitioning in the host plant, that possibly leads to an advantage for the pathogen. Hampering these processes represents a promising strategy to potentially increase resistance. We analyzed the response of the barley monosaccharide transporter HvSTP13 towards biotic stress and its potential use for plant protection. The expression of HvSTP13 increased upon bacterial and fungal PAMP application, suggesting a PAMP-triggered signaling that converged on the transcriptional induction of the gene. Promoter studies indicate a region that is likely targeted by transcription factors downstream of PAMP-triggered immunity pathways. We confirmed that the non-functional HvSTP13GR variant confers resistance against an economically relevant biotrophic rust fungus, in barley. In addition, we established targeted CRISPR/Cas9 cytosine base editing in barley protoplasts to generate alternative HvSTP13 mutants and characterized the sugar transport activity and subcellular localization of the proteins. These mutants represent promising variants for future resistance analysis. Our experimental setup provides basal prerequisites to further decode the role of HvSTP13 in response to biological stress. Moreover, in line with other studies, our experiments indicate that the alteration of sugar partitioning pathways, in a host pathogen interaction, is a promising approach to achieve broad and durable resistance in plants.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation

Background Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. Results We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5′ uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5′ adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. Conclusions We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Genetic Variation in Frost Tolerance, Uromycladium acaciae Rust Resistance, and Growth in an Acacia mearnsii Population

Genetic variation in frost tolerance, resistance to the rust fungus Uromycladium acaciae, growth, stem form, and gummosis were evaluated in 110 open-pollinated families of black wattle (Acacia mearnsii De Wild). Families were tested at six frost-prone sites in northern KwaZulu-Natal and southeastern Mpumalanga, South Africa. Frost-hardy provenances were susceptible to rust disease and had poor growth. Locally grown F1 seed sources that originated from cold-hardy Australian seed sources had better growth and were tolerant to rust, but at a cost of lower frost tolerance. Considerable genetic variation was observed between families within seed sources for frost damage (hfm2 = 0.77), rust incidence (hfm2 = 0.89), and height (hfm2 = 0.80). The corresponding narrow-sense heritabilities (hi2) were 0.30, 0.80, and 0.32. Genotype-by-environment interaction levels were low for most traits. Except for a strong genetic correlation between tree height and diameter (0.90), all the pairs of traits had weak to moderate genetic correlations. Recurrent selection will be successful in improving frost tolerance and rust incidence. However, the current population comprises limited germplasm that is both tolerant to frost and resistant to rust. Thus, we recommend infusing germplasm from known cold-hardy Australian provenances into the current population to increase genetic variation for frost tolerance, rust, and growth. Study Implications Black wattle (Acacia mearnsii De Wild) is widely planted in South Africa for bark extract and woodchip exports. The species is prone to frost damage and susceptible to rust fungus Uromycladium acaciae, two major limitations to its cultivation. In this study, 110 open-pollinated families of black wattle originated from its native range in Australia and local South African seed sources were studied for frost and disease tolerance. The results from this study suggest that there is considerable variation within species for frost tolerance and fungal disease. We suggest a tandem selection strategy for black wattle farming in South Africa to increase resistance to frost damage and fungal diseases.