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[学术文献 ] Auxin resistant 2 and short hypocotyl 2 regulate cotton fiber initiation and elongation 进入全文
PLANT PHYSIOLOGY
Auxin, a pivotal regulator of diverse plant growth processes, remains central to development. The auxin-responsive genes auxin/indole-3-acetic acids (AUX/IAAs) are indispensable for auxin signal transduction, which is achieved through intricate interactions with auxin response factors (ARFs). Despite this, the potential of AUX/IAAs to govern the development of the most fundamental biological unit, the single cell, remains unclear. In this study, we harnessed cotton (Gossypium hirsutum) fiber, a classic model for plant single-cell investigation, to determine the complexities of AUX/IAAs. Our research identified 2 pivotal AUX/IAAs, auxin resistant 2 (GhAXR2) and short hypocotyl 2 (GhSHY2), which exhibit opposite control over fiber development. Notably, suppressing GhAXR2 reduced fiber elongation, while silencing GhSHY2 fostered enhanced fiber elongation. Investigating the mechanistic intricacies, we identified specific interactions between GhAXR2 and GhSHY2 with distinct ARFs. GhAXR2's interaction with GhARF6-1 and GhARF23-2 promoted fiber cell development through direct binding to the AuxRE cis-element in the constitutive triple response 1 promoter, resulting in transcriptional inhibition. In contrast, the interaction of GhSHY2 with GhARF7-1 and GhARF19-1 exerted a negative regulatory effect, inhibiting fiber cell growth by activating the transcription of xyloglucan endotransglucosylase/hydrolase 9 and cinnamate-4-hydroxylase. Thus, our study reveals the intricate regulatory networks surrounding GhAXR2 and GhSHY2, elucidating the complex interplay of multiple ARFs in AUX/IAA-mediated fiber cell growth. This work enhances our understanding of single-cell development and has potential implications for advancing plant growth strategies and agricultural enhancements. Two auxin/indole-3-acetic acid proteins interact with distinct auxin response factors and antagonistically control cotton fiber elongation and initiation.
[学术文献 ] Combined genome and transcriptome analysis of elite fiber quality in Gossypium barbadense 进入全文
PLANT PHYSIOLOGY
Gossypium barbadense, which is one of several species of cotton, is well known for its superior fiber quality. However, the genetic basis of its high-quality fiber remains largely unexplored. Here, we resequenced 269 G. barbadense accessions. Phylogenetic structure analysis showed that the set of accessions was clustered into 3 groups: G1 and G2 mainly included modern cultivars from Xinjiang, China, and G3 was related to widely introduced accessions in different regions worldwide. A genome-wide association study of 5 fiber quality traits across multiple field environments identified a total of 512 qtls (main-effect QTLs) and 94 qtlEs (QTL-by-environment interactions) related to fiber quality, of which 292 qtls and 57 qtlEs colocated with previous studies. We extracted the genes located in these loci and performed expression comparison, local association analysis, and introgression segment identification. The results showed that high expression of hormone-related genes during fiber development, introgressions from Gossypium hirsutum, and the recombination of domesticated elite allelic variation were 3 major contributors to improve the fiber quality of G. barbadense. In total, 839 candidate genes with encoding region variations associated with elite fiber quality were mined. We confirmed that haplotype GB_D03G0092H traced to G. hirsutum introgression, with a 1-bp deletion leading to a frameshift mutation compared with GB_D03G0092B, significantly improved fiber quality. GB_D03G0092H is localized in the plasma membrane, while GB_D03G0092B is in both the nucleus and plasma membrane. Overexpression of GB_D03G0092H in Arabidopsis (Arabidopsis thaliana) significantly improved the elongation of longitudinal cells. Our study systematically reveals the genetic basis of the superior fiber quality of G. barbadense and provides elite segments and gene resources for breeding high-quality cotton cultivars. High expression of hormone-related genes during fiber development, introgressions from Gossypium hirsutum, and domesticated elite allelic variation contribute to the superior fiber quality of Gossypium barbadense.
[学术文献 ] A Verticillium dahliae exoglucanase as potential HIGS target interacts with a cotton cysteine protease to confer resistance to cotton Verticillium wilt 进入全文
PLANT BIOTECHNOLOGY JOURNAL
Verticillium wilt, caused by the soil-borne pathogenic fungus Verticillium dahliae (Vd), represents a devastating disease impacting cotton (Gossypium spp.). However, the limited efficacy of measures to control Verticillium wilt arises because Vd colonizes the host vascular system, as well as the inherent resilience of Vd resting structures (microsclerotia), to various environmental influences (Fradin and Thomma, 2010). Breeding-resistant cotton cultivars are the most economical and efficient approach to increasing host resistance to pathogens (Koch et al., 2019). One such strategy involves the utilization of host-induced gene silencing (HIGS) to target Vd effector genes. We previously employed HIGS to transiently silence the Vd gene encoding an exoglucanase (VdEXG, VDAG_02898) with the typical glycosyl hydrolase family (GH7) domain, which improved host resistance to Vd. However, the underlying molecular mechanisms require further elucidation (Su et al., 2020; Zhao et al., 2015). In this study, we investigated the VdEXG expression pattern in Vd-infected cotton seedlings using reverse transcription quantitative PCR (RT-qPCR). The VdEXG transcript levels increased continuously upon Vd infection and peaked at 12 h post-inoculation (hpi). To elucidate the role of VdEXG in fungal pathogenicity, we knocked out VdEXG in Vd (designated as ΔVdEXG mutant) using a hygromycin resistance cassette by homologous recombination. The penetration capability of ΔVdEXG through a cellophane membrane was notably lower than that of the Vd and ΔVdEXG-complemented (ΔVdEXG-C) strains. Furthermore, ΔVdEXG exhibited substantially reduced growth compared with that of Vd and ΔVdEXG-C strains when cultured in media containing various carbon sources, signifying the indispensable role of VdEXG in vegetative Vd growth. Subsequently, we constructed a recombinant HIGS plasmid targeting the 486-bp VdEXG coding sequences, which was integrated into the cotton genome. This led to the generation of two independent transgenic cotton lines (VdEXG-RNAi-1/2) that displayed heightened resistance to Vd, resulting in decreased fungal biomass compared with that observed in WT. Meanwhile, VdEXG expression at 96 hpi was substantially lower in Vd-infected VdEXG-RNAi transgenic cotton compared with that in WT. Furthermore, siRNA sequencing corroborated the generation of VdEXG-targeting siRNAs in Vd-infected VdEXG-RNAi transgenic cotton. Using RNA hybridization, we observed prominent siVdEXG signals (21–24 nt) in the VdEXG-RNAi lines but not in WT (Figure 1i). These data reveal that the small interfering RNAs (siRNAs) targeting VdEXG reduce the ability of Vd to infect its host and VdEXG as a potential HIGS target to control Vd. Concurrently, fungal glycoside hydrolases are effectors that activate and inhibit host resistance (Cui et al., 2015). SignalP (version 5.0) predicted that VdEXG possesses an N-terminal signal peptide, which was subsequently validated using the yeast signal trap and 2,3,5-triphenyl tetrazolium chloride (TTC) assays. Yeast harbouring the Avr1b effector from Phytophthora sojae and the full-length VdEXG (VdEXGFL) displayed normal growth and caused TTC to turn red, whereas VdEXG lacking the signal peptide sequence (VdEXGNS) and negative controls exhibited no growth and remained colorless. Transient VdEXGNS expression in Nicotiana benthamiana leaves resulted in cell death at 48 hpi, which is consistent with Bcl-2-associated protein X (BAX) rather than eGFP (Cheng et al., 2017). Therefore, we hypothesized that VdEXG functions as an effector to modulate the host immune system. To validate this hypothesis, we identified the cotton cysteine proteinase RD21A (GhRD21A, XM_016851915.2) as a candidate protein interacting with VdEXG from a Vd-inoculated cotton cDNA library. We confirmed the interaction in the yeast two-hybrid (Y2H) assay. Subsequently, we used a bimolecular fluorescence complementation assay in N. benthamiana leaves to verify that VdEXG interacts with RD21A in vivo. Co-expression of VdEXG-nYFP and RD21A-cYFP in plant cells generated a yellow fluorescent signal in the nucleus, indicating the interaction between VdEXG and RD21A. Given the significant reduction in VdEXG expression observed in Vd-infected VdEXG-RNAi cotton lines, we hypothesized that GhRD21A was also inhibited. Concordantly, GhRD21A expression was inhibited in the VdEXG-RNAi lines compared with that in WT at 96 hpi. Furthermore, we ectopically overexpressed GhRD21A in an Arabidopsis ecotype (Col-0) to evaluate its function. The transgenic lines had significantly increased resistance to Vd infection, with reduced necrosis and fungal biomass compared with that in Col-0, which was consistent with the results of a previous study (Zhang et al., 2019). These findings suggest that GhRD21A interacts with VdEXG during Vd infection to promote cotton resistance. In conclusion, our findings suggest that GhRD21A recognized VdEXG to enhance cotton resistance to Vd, while HIGS targeting VdEXG limited the Vd pathogenicity and conferred disease resistance in cotton. These results provide a new strategy for using secretory proteins involved in pathogenicity to breed wilt-resistant cultivars.
[学术文献 ] Subtilisin-like proteases from Fusarium graminearum induce plant cell death and contribute to virulence 进入全文
PLANT PHYSIOLOGY
Fusarium head blight (FHB), caused by Fusarium graminearum, causes huge annual economic losses in cereal production. To successfully colonize host plants, pathogens secrete hundreds of effectors that interfere with plant immunity and facilitate infection. However, the roles of most secreted effectors of F. graminearum in pathogenesis remain unclear. We analyzed the secreted proteins of F. graminearum and identified 255 candidate effector proteins by liquid chromatography-mass spectrometry (LC-MS). Five subtilisin-like family proteases (FgSLPs) were identified that can induce cell death in Nicotiana benthamiana leaves. Further experiments showed that these FgSLPs induced cell death in cotton (Gossypium barbadense) and Arabidopsis (Arabidopsis thaliana). A signal peptide and light were not essential for the cell death-inducing activity of FgSLPs. The I9 inhibitor domain and the entire C-terminus of FgSLPs were indispensable for their self-processing and cell death-inducing activity. FgSLP-induced cell death occurred independent of the plant signal transduction components BRI-ASSOCIATED KINASE 1 (BAK1), SUPPRESSOR OF BIR1 1 (SOBIR1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4). Reduced virulence was observed when FgSLP1 and FgSLP2 were simultaneously knocked out. This study reveals a class of secreted toxic proteins essential for F. graminearum virulence. Five subtilisin-like proteases among the secreted proteins of Fusarium graminearum induce plant cell death and contribute to F. graminearum virulence.
[学术文献 ] Use of Mn3O4 nanozyme to improve cotton salt tolerance 进入全文
PLANT BIOTECHNOLOGY JOURNAL
In this study, we showed that with proper control of size, charge, composition and ROS scavenging ability, the designed PMO (PAA@Mn3O4 nanoparticles) improved salt tolerance in cotton. Also, our previous work showed that the used PMO does not have negative effect on rat heart, liver, spleen, lung and kidney (Shan et al., 2023), suggesting PMO is an environmental friendly and bio-safe nanomaterial. Whether PMO are good candidate to improve abiotic stress tolerance in broad plant species is worthy to be further studied. Our previous work showed that cerium oxide nanoparticles do not enter into the seeds of rice plants (Zhou et al., 2021). Whether PMO will accumulate in seeds or other vegetative parts of plants still needs to be investigated. Moreover, our results showed that Mn3O4 nanoparticles can help to maintain ROS homeostasis and K+/Na+ ratio in cotton plants under salt stress. These newly explored mechanisms for Mn3O4 nanoparticles could give clue to better design Mn-based nanomaterials for nano-enabled agriculture. Overall, our work suggests that learning from nature and designing nanomaterials with desired properties are important factors to facilitate the adoption of nanobiotechnology in agricultural production. Future studies are encouraged to explore more nanomaterials with controlled properties for agricultural applications.
[学术文献 ] Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens 进入全文
NATURE PLANTS
Small RNA (sRNA)-mediated trans-kingdom RNA interference (RNAi) between host and pathogen has been demonstrated and utilized. However, interspecies RNAi in rhizospheric microorganisms remains elusive. In this study, we developed a microbe-induced gene silencing (MIGS) technology by using a rhizospheric beneficial fungus, Trichoderma harzianum, to exploit an RNAi engineering microbe and two soil-borne pathogenic fungi, Verticillium dahliae and Fusarium oxysporum, as RNAi recipients. We first detected the feasibility of MIGS in inducing GFP silencing in V. dahliae. Then by targeting a fungal essential gene, we further demonstrated the effectiveness of MIGS in inhibiting fungal growth and protecting dicotyledon cotton and monocotyledon rice plants against V. dahliae and F. oxysporum. We also showed steerable MIGS specificity based on a selected target sequence. Our data verify interspecies RNAi in rhizospheric fungi and the potential application of MIGS in crop protection. In addition, the in situ propagation of a rhizospheric beneficial microbe would be optimal in ensuring the stability and sustainability of sRNAs, avoiding the use of nanomaterials to carry chemically synthetic sRNAs. Our finding reveals that exploiting MIGS-based biofungicides would offer straightforward design and implementation, without the need of host genetic modification, in crop protection against phytopathogens. This study develops a microbe-induced gene silencing technology by constructing an RNAi engineering microbe to prove the existence of interspecies RNAi in rhizospheric fungi and demonstrate its application in crop protection against fungal pathogens.
