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Alternaria leaf spot seriously threatens the sustainable development of the global apple industry, causing significant losses and reducing fruit quality and yield. The causal agent Alternaria alternata f. sp. mali (Alternaria mali, ALT) produces various molecules to modulate infection, such as cell wall-degrading enzymes, toxins, and elicitor-like molecules. ALT produces the host-specific AM-toxin, an important pathogenicity factor. ALT also releases effectors into apple cells that modify host defense, but these proteins have not yet been described. Here, we identified the pathogenic fungal types responsible for early defoliation from diseased leaves of Fuji (Malus domestica cv. 'Fuji') apple collected from five districts in Shandong Province, China. The ALT isolates ALT2 to ALT7 were pathogenic to four apple cultivars, with ALT7 being the most aggressive. We extracted mycotoxins (AM-toxin-2 to AM-toxin-7) from each isolate and used them to treat different apple varieties, which led to leaf-spot symptoms and damaged chloroplasts and nuclear membranes, followed by cell death. AM-toxin-7 produced the most severe symptoms, but chloroplasts remained intact when the mycotoxin was inactivated. Mass spectrometry identified 134 secretory proteins in ALT7 exosomes, and three secreted proteins (AltABC, AltAO, and AltPDE) were confirmed to be involved in apple pathogenesis. Therefore, ALT secretes AM-toxin and secretory proteins as an infection strategy to promote fungal invasion and overcome the host defense system. [ABSTRACT FROM AUTHOR]

SUMMARY: Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide‐binding, and leucine‐rich repeat domain CCR‐NB‐LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB‐LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1‐inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB‐ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad‐spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple. Significance Statement: Apple leaf spot includes three major fungal diseases affecting the apple industry: Alternaria leaf spot, caused by Alternaria alternata f. sp. mali, Glomerella leaf spot, caused by Glomerella cingulata, and Marssonina leaf spot, caused by Marssonina coronaria. These three devastating diseases of apple (Malus × domestica) cause early severe defoliation, weaken tree vigor, and reduce apple production. In the present study we identified the mode of action of two CCR‐NB‐LRR R proteins, MdRNL2 and MdRNL6, to enhance resistance to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot infection in the resistant apple cultivar HF. The identification and cloning of these novel broad‐spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple. Also, we generated genetically transformed HF plantlets to prove that suppression of MdRNL2 expression results in susceptibility to three apple leaf spot pathogens, similar to the effect of suppressing MdRNL6 in transgenic apple plantlets. Taken together, our data provide detailed evidence for a previously unknown regulatory mechanism in which three apple leaf spot pathogens induce the production of MdRNL2 and MdRNL6, which form a heterocomplex at the NB‐ARC domain and contribute to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot resistance. Our results provide a basis for deciphering the R‐protein‐based defense mechanism against three apple leaf spot pathogens. [ABSTRACT FROM AUTHOR]

Apple leaf spot caused by the Alternaria alternata f. sp mali (ALT1) fungus is one of the most devastating diseases of apple (Malus × domestica). We identified a hairpin RNA (hpRNA) named MdhpRNA277 that produces small RNAs and is induced by ALT1 infection in 'NGR196' apple. MdhpRNA277 produces mdm-siR277-1 and mdm-siR277-2, which target five resistance (R) genes that are expressed at high levels in the resistant apple variety 'Hanfu' and at low levels in the susceptible variety 'NGR196' following ALT1 infection. MdhpRNA277 was strongly induced in 'NGR196' but not 'Hanfu' following ALT1 inoculation. MdhpRNA277 promoter activity was much stronger in inoculated 'NGR196' versus 'Hanfu'. We identified a single-nucleotide polymorphism (SNP) in the MdhpRNA277 promoter region between 'NGR196' (pMdhpRNA277-NGR196) and 'Hanfu' (pMdhpRNA277-HF). The transcription factor MdWHy binds to pMdhpRNA277-NGR196 , but not to pMdhpRNA277-HF. Transgenic 'GL-3' apple expressing pMdhpRNA277-NGR196:MdhpRNA277 was more susceptible to ALT1 infection than plants expressing pMdhpRNA277-HF:MdhpRNA277 due to induced mdm-siR277 accumulation and reduced expression of the five target R genes. We confirmed that the SNP in pMdhpRNA277 is associated with A. alternata leaf spot resistance by crossing. This SNP could be used as a marker to distinguish between apple varieties that are resistant or susceptible to A. alternata leaf spot. [ABSTRACT FROM AUTHOR]

Little is known about the mechanisms by which mRNAs are transported over long distances in the phloem between the rootstock and the scion in grafted woody plants., We identified an mRNA in the pear variety 'Du Li' ( Pyrus betulaefolia) that was shown to be transportable in the phloem. It contains a WUSCHEL- RELATED HOMEOBOX ( WOX) domain and was therefore named Wox Transport 1 ( PbWoxT1)., A 548-bp fragment of PbWoxT1 is critical in long-distance transport. PbWoxT1 is rich in CUCU polypyrimidine domains and its mRNAs interact with a polypyrimidine tract binding protein, Pb PTB3. Furthermore, the expression of PbWoxT1 significantly increased in the stems of wild-type (WT) tobacco grafted onto the rootstocks of PbWoxT1 or Pb PTB3 co-overexpressing lines, but this was not the case in WT plants grafted onto PbWoxT1 overexpressing rootstocks, suggesting that Pb PTB3 mediates PbWoxT1 mRNA long-distance transport., We provide novel information that adds a new mechanism with which to explain the noncell-autonomous manner of WOX gene function, which enriches our understanding of how WOX genes work in fruit trees and other species. [ABSTRACT FROM AUTHOR]

MicroRNA171 (miR171) is a highly conserved miRNA family, crucial for plant growth and development, and has been reported in Arabidopsis thaliana and tomato (Solanum lycopersicum), but the role of miR171 has not been explored in pear. In this study, an effort was made to decipher the mechanism underlying dwarf in ‘Zhongai 3’, of which the shoot length and shoot growth rate during the growing season were much less than those of the vigorous cultivar ‘Zaosu’, and the same for the indole-3-acetic acid (IAA) content in shoot tips after May 22, 2016. We identified a member of the miR171 family, which was most sensitive to IAA and targeted two genes conformed by 5′-RACE, and we named Pyr-miR171f. The two targets were named as PyrSCL6 and PyrSCL22, and contained a GRAS-conserved domain and encoded nucleus proteins. Quantitative RT-PCR analysis revealed that Pyr-miR171f was more abundant in ‘Zaosu’ shoot tips than in ‘Zhongai 3’ shoot tips, whereas the PyrSCL6 and PyrSCL22 mRNAs were more abundant in ‘Zhongai 3’ shoot tips than in ‘Zaosu’ shoot tips. The abundance of Pyr-miR171f and PyrSCL6 and PyrSCL22 mRNAs increased, but the trends were opposite between Pyr-miR171f and its target mRNAs in tissue culture seedlings treated by IAA. Our results suggest that IAA-induced miR171f negatively regulates the IAA signaling cascade via the GRAS pathway to maintain apical dominance. This work reveals a role for the miR171-SCL pathway in the dwarfing of ‘Zhongai 3’, and provides a theoretical basis for dwarf pear breeding. [ABSTRACT FROM AUTHOR]