Biological control of strawberry crown rot,root rot and grey mould by the beneficial fungus Aureobasidium pullulans

Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33–48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.

     

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement,Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.Pomoviruses are causal agents of important diseases affecting potato (Solanum tuberosum), sugar beet (Beta vulgaris), and bean (Phaseolus vulgaris). Potato mop-top virus (PMTV), the type member of the genus Pomovirus, causes an economically important disease of potato called spraing, inducing brown lines and arcs internally and on the surface of tubers. PMTV is transmitted by the root- and tuber-infecting plasmodiophorid Spongospora subterranea (Jones and Harrison, 1969; Arif et al., 1995).The pomovirus genome is divided into three single-stranded RNA (ssRNA) segments of positive polarity. RNA-Rep encodes the putative RNA-dependent RNA polymerase, the replicase of the virus (Savenkov et al., 1999). RNA-CP encodes a coat protein (CP) and another protein called CP-RT or minor CP, which is produced by translational read-through of the CP stop codon (Sandgren et al., 2001). Whereas CP is the major structural protein of the virions, CP-RT is incorporated in one of the termini of the virus particles and a domain within the read-through region of the protein is needed for transmission of the virus by its vector (Reavy et al., 1998). Moreover, CP-RT, but not CP, interacts with the major movement protein TRIPLE GENE BLOCK1 (TGB1; Torrance et al., 2009), which is encoded by RNA-TGB. Besides encoding a triple gene block of movement proteins, TGB1, TGB2, and TGB3 (Zamyatnin et al., 2004), RNA-TGB also encodes a viral suppressor of RNA silencing, the 8K protein (Lukhovitskaya et al., 2013b).To establish a successful infection in the entire plant, viruses must be able to replicate and to move their genomic components between cells, tissues, and organs. Recently, it has become evident that PMTV utilizes a sophisticated mode of cell-to-cell and long-distance movement that involves two virus transport forms, one represented by the viral nucleoprotein complexes (vRNPs) consisting of virus RNA and the TGB1 protein and another represented by the polar virions containing CP-RT and TGB1 proteins attached to one extremity of virus particles (Torrance et al., 2009; for review, see Solovyev and Savenkov, 2014). Proteins implicated in PMTV cell-to-cell movement include TGB1, TGB2, and TGB3 (Zamyatnin et al., 2004; Haupt et al., 2005a). Indirect evidence suggests that CP-RT is required for the efficient systemic movement of intact virions through its interaction with TGB1 (Torrance et al., 2009).Early in infection, the vRNP is transported on the endoplasmic reticulum actomyosin network and targeted to plasmodesmata by TGB2 and TGB3. Later in infection, fluorescently labeled TGB1 is seen in the nucleus and accumulates in the nucleolus. Nucleolar TGB1 association has been shown to be necessary for long-distance movement (Wright et al., 2010).Two structurally distinct subdomains have been identified in the N terminus of TGB1 proteins of hordeiviruses and pomoviruses (Makarov et al., 2009), an N-terminal domain (NTD) comprising approximately 125 amino acids in PMTV (ssRNA in noncooperative and cooperative manners, respectively. The C-terminal half of TGB1 contains a nucleoside triphosphatase/helicase domain that displays cooperative RNA binding. Previously, Wright et al. (2010) reported that TGB1 expressed from a 35S promoter localizes in the cytoplasm and accumulates in the nucleus and nucleolus with occasional labeling of microtubules (MTs). The MT labeling was apparent behind the leading edge of infection when yellow fluorescent protein (YFP)-TGB1 was expressed from an infectious clone. Deletion of 84 amino acids from the N terminus of TGB1 (representing most of the NTD) resulted in the absence of MTs, and nucleolar labeling and fusion of these 84 N-terminal amino acids to GFP resulted in nucleolar enrichment of GFP but no labeling of MTs. Deletion of the 5′ proximal part of the TGB1 open reading frame (ORF), encoding this N-terminal 84 amino acids, in the virus clone abolished systemic but not cell-to-cell movement. However, such deletion had no effect on TGB1 interactions with the CP-RT or self-interaction (Wright et al., 2010).

Table I.

Structural features of the PMTV TGB1 proteinPositively charged amino acids are set in boldface type and underscored. NoD, Nucleolar localization sequence detector; NS, not shown.

Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.