Small RNA Profiling Reveals Phosphorus Deficiency as a Contributing Factor in Symptom Expression for Citrus Huanglongbing Disease

Huanglongbing （HLB） is a devastating citrus disease that is associated with bacteria of the genus ＇Candidatus Liberibacter＇ （Ca. L.）. Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs （sRNA） play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus （Las）-positive or healthy tissue. Ten new microRNAs （miRNAs）, 76 conserved miRNAs, and many small interfering RNAs （siRNAs） were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn--a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

Effective methods for isolation and purification of extracellular vesicles from plants

Plant extracellular vesicles(EVs)play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes,most notably in plant defense responses.However,the isolation of pure,intact EVs from plants remains challenging.A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid(AWF).Here,we compare published plant EV isolation methods,and provide our recommended method for the isolation and purification of plant EVs.This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves,followed by EV isolation via differential centrifugation.To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations,density gradient ultracentrifugation and immunoaffinity capture are then utilized.We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available.Overall,this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.

Bacillus cereus AR156 primes induced systemic resistance by suppressing miR825/825 and activating defense-related genes in Arabidopsis

Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance（ISR） is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here,by comparing small RNA profiles of Pseudomonas syringae pv. tomato（Pst） DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis micro RNAs（mi RNAs） that are differentially regulated by AR156 pretreatment. mi R825 and mi R825 are two mi RNA generated from a single mi RNA gene.Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment. mi R825 targets two ubiquitin-protein ligases,while mi R825 targets toll-interleukin-like receptor（TIR）-nucleotide binding site（NBS） and leucine-rich repeat（LRR）type resistance（R） genes. The expression of these target genes negatively correlated with the expression of mi R825 and mi R825. Moreover, transgenic plants showing reduced expression of mi R825 and mi R825 displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing mi R825 and mi R825 were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing mi R825 and mi R825 and activating the defense related genes they targeted.

BioClay^(TM)prolongs RNA interference-mediated crop protection against Botrytis cinerea

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing(SIGS).In SIGS,small interfering RNA(siRNA)or double-stranded RNA(dsRNA)targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference(RNAi)of the targeted genes,inhibiting fungal growth and disease.However,SIGS is limited by the unstable nature of RNA under environmental conditions.The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA,a formulation termed BioClay^(TM),can enhance RNA durability on plants,prolonging its activity against pathogens.Here,we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea,a major plant fungal pathogen,on tomato leaves and fruit and on mature chickpea plants.BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits,when compared with naked dsRNA.In flowering chickpea plants,BioClay provided prolonged protection for up to 4 weeks,covering the critical period of poding,whereas naked dsRNA provided limited protection.This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.

Mechanisms of Small RNA Generation from Cis-NATs in Response to Environmental and Developmental Cues

ABSTRACT A large proportion of eukaryotic genomes is tranScribed from both positive and negative strands of DNA and thus may generate overlapping sense and antisense transcripts. Some of these so-called natural antisense transcripts （NATs） are possibly co-regulated. When the overlapping sense and antisense transcripts are expressed at the same time in the same cell in response to various developmental and environmental cues; they may form double-stranded RNAs, which could be recognized by the small RNA biogenesis machinery and processed into small interfering RNAs （siRNAs）. cis-NAT-derived siRNAs （nat-siRNAs） are present in plants, animals, and fungi. In plants, the presence of nat-siRNAs is supported not only by Northern blot and genetic analyses, but also by the fact that there is an overall sixfold enrichment of siRNAs in the overlapping regions of cis-NATs and 19%-29% of the siRNA-generating cis-NATs in plants give rise to siRNAs only in their overlapping regions. Silencing mediated by nat-siRNAs is one of the mechanisms for regulating the expression of the cis-NATs. This review focuses on challenging issues related to the biogenesis mechanisms as well as regulation and detection of nat-siRNAs. The advantages and limitations of new technologies for detecting cis-NATs, including direct RNA sequencing and strand-specific RNA sequencing, are also discussed.

Plant biotic interactions

Plants are constantly under attack by pathogens,pests,and parasites,resulting in severe consequences on global food production and human health.While pathogens and pests find their ways to invade and communicate with their hosts,plants have evolved sophisticated immune systems to fight infections.