A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference.

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.

Extracellular RNAs released by plant-associated fungi: from fundamental mechanisms to biotechnological applications.

Extracellular RNAs are an emerging research topic in fungal-plant interactions. Fungal plant pathogens and symbionts release small RNAs that enter host cells to manipulate plant physiology and immunity. This communication via extracellular RNAs between fungi and plants is bidirectional. On the one hand, plants release RNAs encapsulated inside extracellular vesicles as a defense response as well as for intercellular and inter-organismal communication. On the other hand, recent reports suggest that also full-length mRNAs are transported within fungal EVs into plants, and these fungal mRNAs might get translated inside host cells. In this review article, we summarize the current views and fundamental concepts of extracellular RNAs released by plant-associated fungi, and we discuss new strategies to apply extracellular RNAs in crop protection against fungal pathogens. KEY POINTS: • Extracellular RNAs are an emerging topic in plant-fungal communication. • Fungi utilize RNAs to manipulate host plants for colonization. • Extracellular RNAs can be engineered to protect plants against fungal pathogens.

Correction: Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity.

[This corrects the article DOI: 10.1371/journal.ppat.1007288.].

Identification of MaWRKY40 and MaDLO1 as Effective Marker Genes for Tracking the Salicylic Acid-Mediated Immune Response in Bananas.

Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity.

Salicylic acid (SA) is a key phytohormone that mediates a broad spectrum of resistance against a diverse range of viruses; however, the downstream pathway of SA governed antiviral immune response remains largely to be explored. Here, we identified an orchid protein containing A20 and AN1 zinc finger domains, designated Pha13. Pha13 is up-regulated upon virus infection, and the transgenic monocot orchid and dicot Arabidopsis overexpressing orchid Pha13 conferred greater resistance to different viruses. In addition, our data showed that Arabidopsis homolog of Pha13, AtSAP5, is also involved in virus resistance. Pha13 and AtSAP5 are early induced by exogenous SA treatment, and participate in the expression of SA-mediated immune responsive genes, including the master regulator gene of plant immunity, NPR1, as well as NPR1-independent virus defense genes. SA also induced the proteasome degradation of Pha13. Functional domain analysis revealed that AN1 domain of Pha13 is involved in expression of orchid NPR1 through its AN1 domain, whereas dual A20/AN1 domains orchestrated the overall virus resistance. Subcellular localization analysis suggested that Pha13 can be found localized in the nucleus. Self-ubiquitination assay revealed that Pha13 confer E3 ligase activity, and the main E3 ligase activity was mapped to the A20 domain. Identification of Pha13 interacting proteins and substrate by yeast two-hybrid screening revealed mainly ubiquitin proteins. Further detailed biochemical analysis revealed that A20 domain of Pha13 binds to various polyubiquitin chains, suggesting that Pha13 may interact with multiple ubiquitinated proteins. Our findings revealed that Pha13 serves as an important regulatory hub in plant antiviral immunity, and uncover a delicate mode of immune regulation through the coordination of A20 and/or AN1 domains, as well as through the modulation of E3 ligase and ubiquitin chain binding activity of Pha13.

Transcriptome Analysis of Early Defenses in Rice against Fusarium fujikuroi.

BACKGROUND: Bakanae is a seedborne disease caused by Fusarium fujikuroi. Rice seedlings emerging from infected seeds can show diverse symptoms such as elongated and slender stem and leaves, pale coloring, a large leaf angle, stunted growth and even death. Little is known about rice defense mechanisms at early stages of disease development. RESULTS: This study focused on investigating early defenses against F. fujikuroi in a susceptible cultivar, Zerawchanica karatals (ZK), and a resistant cultivar, Tainung 67 (TNG67). Quantitative PCR revealed that F. fujikuroi colonizes the root and stem but not leaf tissues. Illumina sequencing was conducted to analyze the stem transcriptomes of F. fujikuroi-inoculated and mock-inoculated ZK and TNG67 plants collected at 7 days post inoculation (dpi). More differentially expressed genes (DEGs) were identified in ZK (n = 169) than TNG67 (n = 118), and gene ontology terms related to transcription factor activity and phosphorylation were specifically enriched in ZK DEGs. Among the complex phytohormone biosynthesis and signaling pathways, only DEGs involved in the jasmonic acid (JA) signaling pathway were identified. Fourteen DEGs encoding pattern-recognition receptors, transcription factors, and JA signaling pathway components were validated by performing quantitative reverse transcription PCR analysis of individual plants. Significant repression of jasmonate ZIM-domain (JAZ) genes (OsJAZ9, OsJAZ10, and OsJAZ13) at 3 dpi and 7 dpi in both cultivars, indicated the activation of JA signaling during early interactions between rice and F. fujikuroi. Differential expression was not detected for salicylic acid marker genes encoding phenylalanine ammonia-lyase 1 and non-expressor of pathogenesis-related genes 1. Moreover, while MeJA did not affect the viability of F. fujikuroi, MeJA treatment of rice seeds (prior to or after inoculation) alleviated and delayed bakanae disease development in susceptible ZK. CONCLUSIONS: Different from previous transcriptome studies, which analyzed the leaves of infected plants, this study provides insights into defense-related gene expression patterns in F. fujikuroi-colonized rice stem tissues. Twelve out of the 14 selected DEGs were for the first time shown to be associated with disease resistance, and JA-mediated resistance was identified as a crucial component of rice defense against F. fujikuroi. Detailed mechanisms underlying the JA-mediated bakanae resistance and the novel defense-related DEGs are worthy of further investigation.