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<jats:p>Plant diseases cause significant decreases in yield and quality of crops and consequently pose a very substantial threat to food security. In the continuous search for environmentally friendly crop protection, exploitation of RNA interferance machinery is showing promising results. It is well established that small RNAs (sRNAs) including microRNA (miRNA) and small interfering RNA (siRNA) are involved in the regulation of gene expression <jats:italic>via</jats:italic> both transcriptional and post-transcriptional RNA silencing. sRNAs from host plants can enter into pathogen cells during invasion and silence pathogen genes. This process has been exploited through Host-Induced Gene Silencing (HIGS), in which plant transgenes that produce sRNAs are engineered to silence pest and pathogen genes. Similarly, exogenously applied sRNAs can enter pest and pathogen cells, either directly or <jats:italic>via</jats:italic> the hosts, and silence target genes. This process has been exploited in Spray-Induced Gene Silencing (SIGS). Here, we focus on the role of sRNAs and review how they have recently been used against various plant pathogens through HIGS or SIGS-based methods and discuss advantages and drawbacks of these approaches.</jats:p>

<jats:p>Selenium (Se) biofortification is an important strategy for reducing hidden hunger by increasing the nutritional quality of crops. However, there is limited metabolomic information on the nutritional quality of Se-enriched mung beans. In this study, physiological assays and LC–MS/MS based widely targeted metabolomics approach was employed to reveal the Se biofortification potential of mung bean by evaluating the effect of Se on mung bean nutraceutical compounds and their qualitative parameters. Physiological data showed that foliar application of 30 g ha<jats:sup>−1</jats:sup> Se at key growth stages significantly increased the content of Se, protein, fat, total phenols, and total flavonoids content in two mung bean varieties. Widely targeted metabolomics identified 1,080 metabolites, among which L-Alanyl-L-leucine, 9,10-Dihydroxy-12,13-epoxyoctadecanoic acid, and 1-caffeoylquinic acid could serve as biomarkers for identifying highly nutritious mung bean varieties. Pathway enrichment analysis revealed that the metabolic pathways of different metabolites were different in the Se-enriched mung bean. Specifically, P1 was mainly enriched in the linoleic acid metabolic pathway, while P2 was mainly enriched in the phosphonate and phosphinate metabolic pathways. Overall, these results revealed the specific Se enrichment mechanism of different mung bean varieties. This study provides new insights into the comprehensive improvement of the nutritional quality of mung beans.</jats:p>

<jats:p>The hypersensitive response is elicited by <jats:italic>Agrobacterium</jats:italic> infiltration of <jats:italic>Nicotiana benthamiana</jats:italic>, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: <jats:italic>Nb</jats:italic>VPE-1a, <jats:italic>Nb</jats:italic>VPE-1b, and <jats:italic>Nb</jats:italic>CysP6 in the proteolytic degradation of <jats:italic>Nicotiana benthamiana</jats:italic> (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the <jats:italic>NbVPE-1a, NbVPE-1b</jats:italic>, and <jats:italic>NbCysP6</jats:italic> genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the <jats:italic>in vitro</jats:italic> functional activity was detected. Collectively, we demonstrate an innovative <jats:italic>in planta</jats:italic> strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.</jats:p>

<jats:p>Mechanical strength is essential for the upright growth habit, which is one of the most important characteristics of terrestrial plants. Lignin, a phenylpropanoid-derived polymer mainly present in secondary cell walls plays critical role in providing mechanical support. Here, we report that the prostrate-stem cultivar of the legume forage <jats:italic>Medicago ruthenica</jats:italic> cultivar ‘Mengnong No. 1’ shows compromised mechanical strength compared with the erect-stem cultivar ‘Zhilixing’. The erect-stem cultivar, ‘Zhilixing’ has significantly higher lignin content, leading to higher mechanical strength than the prostrate-stem cultivar. The low abundance of miRNA397a in the Zhiixing cultivar causes reduced cleavage of <jats:italic>MrLAC17</jats:italic> transcript, which results in enhanced expression level of <jats:italic>MrLAC17</jats:italic> compared to that in the prostrate-stem cultivar Mengnong No. 1. Complementation of the <jats:italic>Arabidopsis lac4 lac17</jats:italic> double mutants with <jats:italic>MrLAC17</jats:italic> restored the lignin content to wild-type levels, confirming that MrLAC17 perform an exchangeable role with <jats:italic>Arabidopsis</jats:italic> laccases. <jats:italic>LAC17-</jats:italic>mediated lignin polymerization is therefore increased in the ‘Zhilixing’, causing the erect stem phenotype. Our data reveal the importance of the miR397a in the lignin biosynthesis and suggest a strategy for molecular breeding targeting plant architecture in legume forage.</jats:p>

<jats:p><jats:italic>Fusarium oxysporum</jats:italic> is a main causative agent of tobacco root rot, severely affecting tobacco growth. Here, 200 <jats:italic>F. oxysporum</jats:italic> strains were isolated and examined for their virulence toward tobacco plants. These strains were divided into disease class 1–3 (weak virulence), 4–6 (moderate virulence), and 7–9 (strong virulence). To understand the virulence mechanism of <jats:italic>F. oxysporum</jats:italic>, a comparative transcriptome study was performed using weak, moderate, and strong virulence-inducing strains. The results showed that expression levels of 1,678 tobacco genes were positively correlated with virulence levels, while expression levels of 3,558 genes were negatively associated with virulence levels. Interestingly, the expression levels of ATP synthase genes were positively correlated with <jats:italic>F. oxysporum</jats:italic> virulence. To verify whether ATP synthase gene expression is associated with <jats:italic>F. oxysporum</jats:italic> virulence, 5 strains each of strong, moderate, and weak virulence-inducing strains were tested using qRT-PCR. The results confirmed that ATP synthase gene expression is positively correlated with virulence levels. Knock-out mutants of ATP synthase genes resulted in a relatively weak virulence compared to wild-type as well as the inhibition of <jats:italic>F. oxysporum</jats:italic>-mediated suppression of <jats:italic>NtSUC4</jats:italic>, <jats:italic>NtSTP12</jats:italic>, <jats:italic>NtHEX6</jats:italic>, and <jats:italic>NtSWEET</jats:italic>, suggesting that ATP synthase activity is also associated with the virulence. Taken together, our analyses show that ATP synthases are key genes for the regulation of <jats:italic>F. oxysporum</jats:italic> virulence and provide important information for understanding the virulence mechanism of <jats:italic>F. oxysporum</jats:italic> in tobacco root rot.</jats:p>

<jats:p>Calmodulin (CaM) and calmodulin-like (CML) proteins are Ca<jats:sup>2+</jats:sup> relays and play diverse and multiple roles in plant growth, development and stress responses. However, <jats:italic>CaM/CML</jats:italic> gene family has not been identified in barley (<jats:italic>Hordeum vulgare</jats:italic>). In the present study, 5 <jats:italic>HvCaMs</jats:italic> and 80 <jats:italic>HvCMLs</jats:italic> were identified through a genome-wide analysis. All HvCaM proteins possessed 4 EF-hand motifs, whereas HvCMLs contained 1 to 4 EF-hand motifs. <jats:italic>HvCaM2</jats:italic>, <jats:italic>HvCaM3</jats:italic> and <jats:italic>HvCaM5</jats:italic> coded the same polypeptide although they differed in nucleotide sequence, which was identical to the polypeptides coded by <jats:italic>OsCaM1-1</jats:italic>, <jats:italic>OsCaM1-2</jats:italic> and <jats:italic>OsCaM1-3</jats:italic>. <jats:italic>HvCaMs/CMLs</jats:italic> were unevenly distributed over barley 7 chromosomes, and could be phylogenetically classified into 8 groups. <jats:italic>HvCaMs/CMLs</jats:italic> differed in gene structure, <jats:italic>cis</jats:italic>-acting elements and tissue expression patterns. Segmental and tandem duplication were observed among <jats:italic>HvCaMs/CMLs</jats:italic> during evolution. <jats:italic>HvCML16</jats:italic>, <jats:italic>HvCML18</jats:italic>, <jats:italic>HvCML50</jats:italic> and <jats:italic>HvCML78</jats:italic> were dispensable genes and the others were core genes in barley pan-genome. In addition, 14 <jats:italic>HvCaM/CML</jats:italic> genes were selected to examine their responses to salt, osmotic and low potassium stresses by qRT-PCR, and their expression were stress-and time-dependent. These results facilitate our understanding and further functional identification of <jats:italic>HvCaMs/CMLs</jats:italic>.</jats:p>

<jats:p>Chickpea is a cool season crop that is highly vulnerable to abiotic stresses such as heat and drought. High temperature during early flowering and pod development stages significantly reduces the crop yield. The wild relatives of chickpeas can be potential donors for the introgression of heat and drought tolerance into cultivated chickpeas for crop improvement. Initially, 600 interspecific lines were derived from crosses between two elite cultivars, CDC Leader (kabuli chickpea) and CDC Consul (desi chickpea), and 20 accessions of <jats:italic>Cicer reticulatum</jats:italic>. The F<jats:sub>5</jats:sub> interspecific lines were tested for agronomic and seed quality traits including reaction to ascochyta blight disease under field conditions at two locations in 2018. A subset of 195 lines were selected based on resistance to ascochyta blight and acceptable seed quality. These lines were evaluated for their performance under suboptimal conditions at Lucky Lake (2019 and 2020) and Moose Jaw (2019), Saskatchewan, Canada, and Yuma, Arizona, United States (2019–2020). The lines were grown and evaluated at two seeding dates, normal (SD1) and late (SD2) seeding dates, at each location and year. The same lines were genotyped using Cicer60K Axiom® SNP chip. The population structure was determined based on 35,431 informative SNPs using fastStructure, and the interspecific lines were clustered at a <jats:italic>k</jats:italic>-value of 15. Significant marker-trait associations were identified for seed yield from SD1 and SD2 seeding dates, and stress tolerance indices (ATI, K<jats:sub>1</jats:sub>STI, MP, SSPI, and TOL) using phenotypic values both from individual locations and combined analyses based on BLUP values. SNP marker Ca2_34600347 was significantly associated with yield from both the seeding dates. This and other SNP markers identified in this study may be useful for marker-assisted introgression of abiotic stress tolerance in chickpea.</jats:p>

<jats:p>One of the greatest threats to wild strawberries (<jats:italic>Fragaria vesca</jats:italic> Mara des Bois) after harvest is the highly perishability at ambient temperature. Breeders have successfully met the quality demands of consumers, but the prevention of waste after harvest in fleshy fruits is still pending. Most of the waste is due to the accelerated progress of senescence-like process after harvest linked to a rapid loss of water and firmness at ambient temperature. The storage life of strawberries increases at low temperature, but their quality is limited by the loss of cell structure. The application of high CO<jats:sub>2</jats:sub> concentrations increased firmness during cold storage. However, the key genes related to resistance to softening and cell wall disassembly following transference from cold storage at 20°C remain unclear. Therefore, we performed RNA-seq analysis, constructing a weighted gene co-expression network analysis (WGCNA) to identify which molecular determinants play a role in cell wall integrity, using strawberries with contrasting storage conditions, CO<jats:sub>2</jats:sub>-cold stored (CCS), air-cold stored (ACS), non-cold stored (NCS) kept at ambient temperature, and intact fruit at harvest (AH). The hub genes associated with the cell wall structural architecture of firmer CO<jats:sub>2</jats:sub>-treated strawberries revealed xyloglucans stabilization attributed mainly to a down-regulation of <jats:italic>Csl E1</jats:italic>, <jats:italic>XTH 15</jats:italic>, <jats:italic>Exp-like B1</jats:italic> and the maintenance of expression levels of nucleotide sugars transferases such as <jats:italic>GMP</jats:italic> and <jats:italic>FUT</jats:italic> as well as improved lamella integrity linked to a down-regulation of <jats:italic>RG-lyase</jats:italic>, <jats:italic>PL-like</jats:italic> and <jats:italic>PME</jats:italic>. The preservation of cell wall elasticity together with the up-regulation of <jats:italic>LEA</jats:italic>, <jats:italic>EXPA4</jats:italic>, and <jats:italic>MATE</jats:italic>, required to maintain cell turgor, is the mechanisms controlled by high CO<jats:sub>2</jats:sub>. In stressed air-cold stored strawberries, in addition to an acute softening, there is a preferential transcript accumulation of genes involved in lignin and raffinose pathways. Non-cold stored strawberries kept at 20°C after harvest are characterized by an enrichment in genes mainly involved in oxidative stress and up-expression of genes involved in jasmonate biosynthesis. The present results on transcriptomic analysis of CO<jats:sub>2</jats:sub>-treated strawberries with enhanced resistance to softening and oxidative stress at consumption will help to improve breeding strategies of both wild and cultivated strawberries.</jats:p>

<jats:p>The HD-Zip transcription factors play a crucial role in plant development, secondary metabolism, and abiotic stress responses, but little is known about HD-Zip I genes in soybean. Here, a homeodomain-leucine zipper gene designated <jats:italic>GmHdz4</jats:italic> was isolated. Chimeric soybean plants, <jats:italic>GmHdz4</jats:italic> overexpressing (<jats:italic>GmHdz4-oe</jats:italic>), and gene-editing <jats:italic>via</jats:italic> CRISPR/Cas9 (<jats:italic>gmhdz4</jats:italic>) in hairy roots, were generated to examine the <jats:italic>GmHdz4</jats:italic> gene response to polyethylene glycol (PEG)-simulated drought stress. Bioinformatic analysis showed <jats:italic>GmHdz4</jats:italic> belonged to clade δ, and was closely related to other drought tolerance-related HD-Zip I family genes such as <jats:italic>AtHB12</jats:italic>, <jats:italic>Oshox12</jats:italic>, and <jats:italic>Gshdz4</jats:italic>. The <jats:italic>GmHdz4</jats:italic> was located in the plant nucleus and showed transcriptional activation activity by yeast hybrid assay. Quantitative real-time PCR analysis revealed that <jats:italic>GmHdz4</jats:italic> expression varied in tissues and was induced by PEG-simulated drought stress. The <jats:italic>gmhdz4</jats:italic> showed promoted growth of aboveground parts, and its root system architecture, including the total root length, the root superficial area, and the number of root tips were significantly higher than those of <jats:italic>GmHdz4-oe</jats:italic> even the non-transgenic line (NT) on root tips number. The better maintenance of turgor pressure by osmolyte accumulation, and the higher activity of antioxidant enzymes to scavenge reactive oxygen species, ultimately suppressed the accumulation of hydrogen peroxide (H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>), superoxide anion (O<jats:sup>2−</jats:sup>), and malondialdehyde (MDA), conferring higher drought tolerance in <jats:italic>gmhdz4</jats:italic> compared with both <jats:italic>GmHdz4-oe</jats:italic> and NT. Together, our results provide new insights for future research on the mechanisms by which <jats:italic>GmHdz4</jats:italic> gene-editing <jats:italic>via</jats:italic> CRISPR/Cas9 system could promote drought stress and provide a potential target for molecular breeding in soybean.</jats:p>

<jats:p>Weeping cypress is an endemic tree species that is widely planted in China, and the simple stand structure and fragile ecosystem of its plantation are common issues. Exploring the effect of different gap sizes on the soil bacterial community structure of weeping cypress plantations can provide a theoretical basis for the near-natural management of forest plantations. We, therefore, constructed three kinds of forest gaps with different sizes in weeping cypress plantations, namely, small (50–100 m<jats:sup>2</jats:sup>), medium (100–200 m<jats:sup>2</jats:sup>), and large gaps (400–667 m<jats:sup>2</jats:sup>), for identifying the key factors that affect soil bacterial communities following the construction of forest gaps. The results suggested that the herb layer was more sensitive than the shrub layer, while the Simpson, Shannon, and richness indices of the herb layer in plots with gaps were significantly higher than those of designated sampling plots without any gaps (CK). The presence of large gaps significantly increased the understory plant diversity and the Shannon and Simpson indices of the soil bacterial alpha diversity. There were obvious changes in the community composition of soil bacteria following the construction of forest gaps. The dominant bacterial phyla, orders, and functions were similar across the plots with different gap sizes. Of the indicator bacterial species, the abundance of the nitrogen-fixing bacteria, <jats:italic>Lysobacter_ yangpyeongensis</jats:italic>, and <jats:italic>Ensifer_meliloti</jats:italic>, was significantly different across plots with different gap sizes and accounted for a large proportion of the bacterial population of plots with medium and large gaps. The understory plant diversity was mostly related to the soil bacterial community than to other soil factors. The results of structural equation modeling indicated that the understory plant diversity was the most important environmental factor in driving the composition and diversity of bacterial communities. The construction of forest gaps significantly improved the understory plant diversity, physicochemical properties of the soil, and bacterial diversity in weeping cypress plantations, and the results of the comprehensive evaluation were in the order: large gaps &amp;gt; small gaps &amp;gt; medium gaps &amp;gt; CK. Our results suggested that large gaps are beneficial for the diversity of above-ground plant communities and underground soil bacterial communities.</jats:p>