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Magnaporthe oryzae is the causal agent of rice blast disease, a major threat to global food security. Although M. oryzae infects a broad range of monocotyledonous plants, it fails to colonize dicot species such as Nicotiana benthamiana, offering a useful system to investigate nonhost resistance (NHR). In this study, we characterized the immune responses of N. benthamiana to M. oryzae by profiling defense-related gene expression, analyzing fungal invasion, and functionally dissecting key immune components. Time-course expression analyses revealed sustained upregulation of NbBAK1, NbEAS, NbWRKY22, and NbPR1, alongside dynamic regulation of NbCYP71D20 and NbSGT1. Virus-induced gene silencing demonstrated that silencing of NbSGT1, but not NbEAS or NbBAK1, significantly enhanced fungal colonization. Furthermore, salicylic acid (SA)-deficient NahG plants exhibited increased susceptibility, suggesting that SA and SGT1-dependent immunity synergistically contribute to NHR. Visualization of infection using a GFP-expressing fungal strain confirmed that suppression of SGT1 and SA signaling facilitated hyphal expansion into adjacent host cells. High-throughput screening of 179 M. oryzae candidate effectors revealed that 70 induced hypersensitive response-like cell death in N. benthamiana, a response that was abrogated by NbSGT1 silencing. These findings collectively demonstrate that SA signaling and SGT1-dependent effector-triggered immunity are critical barriers against M. oryzae invasion and highlight the potential of nonhost immune components as resources for engineering durable resistance in crops. 

The common rust disease of maize is caused by the obligate biotrophic fungusPuccinia sorghi. The maizeRp1-Dallele imparts resistance against theP.sorghiIN2 isolate by initiating a defense response that includes a rapid localized programmed cell death process, the hypersensitive response (HR). In this study, to identify AvrRp1-D fromP.sorghiIN2, we employed the isolation of haustoria, facilitated by a biotin-streptavidin interaction, as a powerful approach. This method proves particularly advantageous in cases where the genome information for the fungal pathogen is unavailable, enhancing our ability to explore and understand the molecular interactions between maize andP.sorghi. The haustorial transcriptome generated through this technique, in combination with bioinformatic analyses such as SignalP and TMHMM, enabled the identification of 251 candidate effectors. We ultimately identified two closely related genes,AvrRp1-D.1andAvrRp1-D.2, which triggered anRp1-D-dependent defense response inNicotiana benthamiana.AvrRp1-D-inducedRp1-D-dependent HR was further confirmed in maize protoplasts. We demonstrated that AvrRp1-D.1 interacts directly and specifically with the leucine-rich repeat (LRR) domain of Rp1-D through yeast two-hybrid assay. We also provide evidence that, in the absence of Rp1-D, AvrRp1-D.1 plays a role in suppressing the plant immune response. Our research provides valuable insights into the molecular interactions driving resistance against common rust in maize. 

Haplotype-level allelic characterization facilitates research on the functional, evolutionary and breeding-related features of extremely large and complex plant genomes. We report a 21.7-Gb chromosome-level haplotype-resolved assembly in Pinus densiflora. We found genome rearrangements involving translocations and inversions between chromosomes 1 and 3 of Pinus species and a proliferation of specific long terminal repeat (LTR) retrotransposons (LTR-RTs) in P. densiflora. Evolutionary analyses illustrated that tandem and LTR-RT-mediated duplications led to an increment of transcription factor (TF) genes in P. densiflora. The haplotype sequence comparison showed allelic imbalances, including presence–absence variations of genes (PAV genes) and their functional contributions to flowering and abiotic stress-related traits in P. densiflora. Allele-aware resequencing analysis revealed PAV gene diversity across P. densiflora accessions. Our study provides insights into key mechanisms underlying the evolution of genome structure, LTR-RTs and TFs within the Pinus lineage as well as allelic imbalances and diversity across P. densiflora. 

Abstract Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)−1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogensBotrytis cinereaandMagnaporthe oryzaeATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation inAscomycotapathogens,B. cinerea, M. oryzae,Sclerotinia sclerotiorumandMonilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens. 

Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastid–nuclear complexes, which provide close physical contact between these organelles. Plastid-nuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H 2 O 2 to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid–nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid–nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state, the two modes of connection contribute to varying extents. We hypothesize that this association is dynamic and that there may be a link between plastid–nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.