Ustilaginoidea virens , the causal agent of rice false smut (RFS), has become one of the most devastating rice pathogens worldwide. As a group of essential virulence factors, the effectors in the filamentous fungus might play central roles in the interaction between plants and pathogens. However, little is known about the roles of individual effectors in U. virens virulence. In this study, we identified and characterized a small secreted cysteine-rich effector, SCRE2, in U. virens . SCRE2 was first confirmed as an effector through yeast secretion, protein localization and translocation assays, as well as its expression pattern during U. virens infection. Transient expression of SCRE2 in Nicotiana benthamiana suppressed necrosis-like defense symptoms triggered by the mammalian BAX and oomycete elicitin INF1 proteins. The ability of SCRE2 to inhibit immunity-associated responses in N. benthamiana , including elicitor-triggered cell death and oxidative burst, is further defined to a small peptide region SCRE2 68-85 through expressing a series of truncated proteins. Convincingly, ectopic expression of SCRE2 in the transgenic rice cells significantly inhibited pathogen-associated molecular pattern-triggered immunity including flg22- and chitin-induced defense gene expression and oxidative burst. Furthermore, the scre2 knockout mutant generated by the CRISPR/Cas9 system greatly attenuated in U. virens virulence to rice. Collectively, this study indicates that the effector SCRE2 is able to inhibit plant immunity and is required for full virulence of U. virens .

The WRKY web, which is comprised of a subset of WRKY transcription factors (TFs), plays a crucial role in the regulation of plant immunity, however, the mode of organization and operation of this network remains obscure, especially in non-model plants such as pepper (Capsicum annuum). Herein, CaWRKY22, a member of a subgroup of IIe WRKY proteins from pepper, was functionally characterized in pepper immunity against Ralstonia Solanacearum. CaWRKY22 was found to target the nuclei, and its transcript level was significantly upregulated by Ralstonia Solanacearum inoculation (RSI) and exogenously applied salicylic acid (SA), Methyl jasmonate (MeJA), or ethephon (ETH). Loss-of-function CaWRKY22, caused by virus-induced gene silencing (VIGS), enhanced pepper’s susceptibility to RSI. In addition, the silencing of CaWRKY22 perturbed the hypersensitive response (HR)-like cell death elicited by RSI and downregulated defense-related genes including CaPO2, CaPR4, CaACC, CaBPR1, CaDEF1, CaHIR1, and CaWRKY40. CaWRKY22 was found to directly bind to the promoters of CaPR1, CaDEF1, and CaWRKY40 by chromatin immuno-precipitation (ChIP) analysis. Contrastingly, transient overexpression of CaWRKY22 in pepper leaves triggered significant HR-like cell death and upregulated the tested immunity associated maker genes. Moreover, the transient overexpression of CaWRKY22 upregulated the expression of CaWRKY6 and CaWRKY27 while it downregulated of the expression of CaWRKY58. Conversely, the transient overexpression of CaWRKY6, CaWRKY27, and CaWRKY40 upregulated the expression of CaWRKY22, while transient overexpression of CaWRKY58 downregulated the transcript levels of CaWRKY22. These data collectively recommend the role of CaWRKY22 as a positive regulator of pepper immunity against R. Solanacearum, which is regulated by signaling synergistically mediated by SA, jasmonic acid (JA), and ethylene (ET), integrating into WRKY networks with WRKY TFs including CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58.

Phytopathogenic fungi secrete a large arsenal of effector molecules, including proteinaceous effectors, small RNAs, phytohormones and derivatives thereof. The pathogenicity of fungal pathogens is primarily determined by these effectors that are secreted into host cells to undermine innate immunity, as well as to facilitate the acquisition of nutrients for their in planta growth and proliferation. After conventional and non-conventional secretion, fungal effectors are translocated into different subcellular compartments of the host cells to interfere with various biological processes. In extracellular spaces, apoplastic effectors cope with physical and chemical barriers to break the first line of plant defenses. Intracellular effectors target essential immune components on the plasma membrane, in the cytosol, including cytosolic organelles, and in the nucleus to suppress host immunity and reprogram host physiology, favoring pathogen colonization. In this review, we comprehensively summarize the recent advances in fungal effector biology, with a focus on the versatile virulence functions of fungal effectors in promoting pathogen infection and colonization. A perspective of future research on fungal effector biology is also discussed.

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Rice false smut caused by Ustilaginoidea virens is emerging as a devastating disease of rice (Oryza sativa) worldwide; however, the molecular mechanisms underlying U. virens virulence and pathogenicity remain largely unknown. Here we demonstrate that the small cysteine-rich secreted protein SCRE6 in U. virens is translocated into host cells during infection as a virulence factor. Knockout of SCRE6 leads to attenuated U. virens virulence to rice. SCRE6 and its homologs in U. virens function as a novel family of mitogen-activated protein (MAP) kinase phosphatases harboring no canonical phosphatase motif. SCRE6 interacts with and dephosphorylates the negative immune regulator OsMPK6 in rice, thus enhancing its stability and suppressing plant immunity. Ectopic expression of SCRE6 in transgenic rice promotes pathogen infection by suppressing the host immune responses. Our results reveal a previously unidentified fungal infection strategy in which the pathogen deploys a family of tyrosine phosphatases to stabilize a negative immune regulator in the host plant to facilitate its infection.