Plants utilize extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called “cross-kingdom RNAi.” It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins (RBPs) in Arabidopsis , including Argonaute 1 (AGO1), RNA helicases (RHs) and Annexins (ANN), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not non-EV enriched sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11rh37 and ann1ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RBPs play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11rh37 and ann1ann2 showed increased susceptibility to Botrytis cinerea , supporting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea .

Eukaryotic small RNAs (sRNAs) are short non-coding regulatory molecules that induce RNA interference (RNAi). During microbial infection, host RNAi machinery is highly regulated and contributes to reprogramming gene expression and balancing plant immunity and growth. While most sRNAs function endogenously, some can travel across organismal boundaries between hosts and microbes and silence genes in trans in interacting organisms, a mechanism called ''cross-kingdom RNAi.'' During the co-evolutionary arms race between fungi and plants, some fungi developed a novel virulence mechanism, sending sRNAs as effector molecules into plant cells to silence plant immunity genes, whereas plants also transport sRNAs, mainly using extracellular vesicles, into the pathogens to suppress virulence-related genes. In this Review, we highlight recent discoveries on these key roles of sRNAs and RNAi machinery. Understanding the molecular mechanisms of sRNA biogenesis, trafficking, and RNAi machinery will help us develop innovative strategies for crop protection.

Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs.Eukaryotes and prokaryotes contain neutral lipids in subcellular droplets as food reserves and/or for other purposes (Hsieh and Huang, 2004;Martin and Parton, 2006;Goodman, 2008;Rajakumari et al., 2008). These lipid droplets are present in seeds, pollens, fruits, and flowers of higher plants; the vegetative and reproductive organs of lower plants, algae, fungi, and nematodes; mammalian organs/tissues, such as mammalian glands and adipose tissues; and bacteria. Among all these lipid droplets, oil bodies (OBs) in seeds are the most prominent and have been extensively studied.Seeds of diverse plant species store oils (triacylglycerols [TAGs]) as food reserves for germination and postgermination growth (Napier et al., 1996;Frandsen et al., 2001;Murphy, 2001;Hsieh and Huang, 2004).The TAGs are present in small subcellular, spherical OBs of approximately 0.5 to 2 mm in diameter. Each OB has a matrix of TAGs surrounded by a layer of phospholipids (PLs) and the structural protein oleosins. The massive oleosins completely cover the surface of the OBs and prevent them from coalescence; so, a large surface area per unit TAG is available for lipase binding and catalysis during germination. Each oleosin molecule has a characteristic long central hydrophobic stretch, which forms a hairpin penetrating into the matrix TAGs for stable anchorage.Other than being present in the seeds of plants, oleos...

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr. Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage, and transfer of these pollen-coat materials in maize (Zea mays) differs completely from that reported in Arabidopsis (Arabidopsis thaliana), which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, three proteins, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease (ZmPCP), are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid developmental stage, whereas protease transcript emerged at a late developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanase transcript was distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative amino-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All three proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid to late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the three proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or the pollen surface.