Loss-of-function alleles of ZmPLD3 cause haploid induction in maize

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

Mcc1229, an Stx2a-amplifying microcin, is produced in vivo and requires CirA for activity

Enterohemorrhagic E. coli (EHEC) strains, including the foodborne pathogen E. coli O157:H7, are responsible for thousands of hospitalizations each year. Various environmental triggers can modulate pathogenicity in EHEC by inducing expression of Shiga toxin (Stx), which is encoded on a lambdoid prophage and transcribed together with phage late genes. Cell-free supernatants of the sequence type (ST) 73 E. coli strain 0.1229 are potent inducers of Stx2a production in EHEC, suggesting that 0.1229 secretes a factor that activates the SOS response and leads to phage lysis. We previously demonstrated that this factor, designated microcin (Mcc) 1229, was proteinaceous and plasmid-encoded. To further characterize Mcc1229 and support its classification as a microcin, we investigated its regulation, determined its receptor, and identified loci providing immunity. Production of Mcc1229 was increased upon iron limitation, as determined by ELISA, lacZ fusions, and qRT-PCR. Spontaneous Mcc1229-resistant mutants and targeted gene deletion revealed that CirA was the Mcc1229 receptor. TonB, which interacts with CirA in the periplasm, was also essential for Mcc1229 import. Subcloning of the Mcc1229 plasmid indicated that Mcc activity was neutralized by two ORFs, each predicted to encode a domain of unknown function (DUF)-containing protein. In a germfree mouse model of infection, colonization with 0.1229 suppressed subsequent colonization of EHEC. Although Mcc1229 was produced in vivo , it was dispensable for colonization suppression. The regulation, import, and immunity determinants identified here are consistent with features of other Mccs, suggesting that Mcc1229 be included in this class of small molecules.

Identification and Expression Analysis of DUF4228 Domain Containing (DDP) Genes in Potato Under Abiotic and Phytohormone Stress

DUF4228 (Domain of unknown function 4228) proteins are widely distributed in plants and performed a vital role in abiotic stress response. In potato (Solanum tuberosum), the study of the DUF4228 gene family is sparse. To understant the role of DUF4228 in potato a comprehensive genome-wide analysis was carried out in potato genome. Further, the StDUAF4228 genes relative expression was also evaluated in various plant tissues. In the present study identified 31 StDUF4228 genes were clustered into six groups. The promoter analysis of StDUF4228 revealed various cis-acting regulatory components response to abiotic and phytohormones stresses. Under selection pressure, gene duplication also showed various positive and purifying selections in StDUF4228 genes. Expression profiling indicated that StDUF4228 genes were broadly expressed in various tissues and organs in potato. Moreover, the StDUF4228 genes profiles expression was characterized through RNA-sequence analysis data under salt and heat treatments. In potato, StDUF4228-4, StDUF4228-14, StDUF4228-21, and StDUF4228-30 represented a high expression under salt and heat stress.Furthermore, 23 genes under IAA treatment and 18 under ABA treatment, showed that IAA and ABA played a vital role in stresses. The RT-qPCR and expression profilling exhibited high expression in root tissues. Moreover multiple miRNA target StDUF4228 genes in potato.These expression results provide primary reference data and functional analysis data for other commercial crops.

A novel Calmodulin-interacting Domain of Unknown Function 506 protein represses root hair elongation in Arabidopsis

DUF506 proteins are omnipresent in higher plants. Phosphorus (P) stress-inducible AtRXR1/REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 gene, as the first functionally characterized DUF506 gene, is proved to inhibit root hair elongation through interaction of RXR2/RabD2c GTPase. However, the knowledge of other P-responsive DUF506 genes is still limited. Here, we identify four additional P-inducible DUF506 genes and select two of candidates for functional investigation. Expression analysis results reveal that both of candidates are induced by auxin. At3g07350, the duplicated pair of RXR1, expresses ubiquitously in seedlings under P-stress, whereas At1g62420 is mainly induced in roots. Overexpression and knockout mutants of At1g62420, which is called RXR3, exhibit shorter or longer root hair length, respectively. Cellular imaging results demonstrate RXR3 localizes in root epidermal cells. ChIP, synthetic peptide treatment and qRT-PCR assay results indicate RXR3 is transcriptionally activated by RSL4 or RALF1. BiFC and CaM-binding assay suggest that RXR3 interacts with various CaMs in presence of Ca2+. Moreover, the frequencies of [Ca2+]cyt in rxr3 mutants are approximately 20% higher compared to that of wild type. Taken together, our results illustrate a divergent mechanism by which RSL4-directed RXR3 interacts with calmodulin and functions in parallel of RXR1, to prevent root hair excessive growth.

The citron homology domain as a scaffold for Rho1 signaling

Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate β-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed β-propeller fold containing three unusual loops. A model of the Rho1–Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis.

Comparative Genomic Analysis of the DUF34 Protein Family Suggests Role as a Metal Ion Chaperone or Insertase

Members of the DUF34 (domain of unknown function 34) family, also known as the NIF3 protein superfamily, are ubiquitous across superkingdoms. Proteins of this family have been widely annotated as “GTP cyclohydrolase I type 2” through electronic propagation based on one study. Here, the annotation status of this protein family was examined through a comprehensive literature review and integrative bioinformatic analyses that revealed varied pleiotropic associations and phenotypes. This analysis combined with functional complementation studies strongly challenges the current annotation and suggests that DUF34 family members may serve as metal ion insertases, chaperones, or metallocofactor maturases. This general molecular function could explain how DUF34 subgroups participate in highly diversified pathways such as cell differentiation, metal ion homeostasis, pathogen virulence, redox, and universal stress responses.

Evolutionary and Characteristic Analysis of RING-DUF1117 E3 Ubiquitin Ligase Genes in Gossypium Discerning the Role of GhRDUF4D in Verticillium dahliae Resistance

Verticillium wilt, primarily induced by the soil-borne fungus Verticillium dahliae, is a serious threat to cotton fiber production. There are a large number of really interesting new gene (RING) domain-containing E3 ubiquitin ligases in Arabidopsis, of which three (At2g39720 (AtRHC2A), At3g46620 (AtRDUF1), and At5g59550 (AtRDUF2)) have a domain of unknown function (DUF) 1117 domain in their C-terminal regions. This study aimed to detect and characterize the RDUF members in cotton, to gain an insight into their roles in cotton’s adaptation to environmental stressors. In this study, a total of 6, 7, 14, and 14 RDUF (RING-DUF1117) genes were detected in Gossypium arboretum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These RDUF genes were classified into three groups. The genes in each group were highly conserved based on gene structure and domain analysis. Gene duplication analysis revealed that segmental duplication occurred during cotton evolution. Expression analysis revealed that the GhRDUF genes were widely expressed during cotton growth and under abiotic stresses. Many cis-elements related to hormone response and environment stressors were identified in GhRDUF promoters. The predicted target miRNAs and transcription factors implied that GhRDUFs might be regulated by gra-miR482c, as well as by transcription factors, including MYB, C2H2, and Dof. The GhRDUF genes responded to cold, drought, and salt stress and were sensitive to jasmonic acid, salicylic acid, and ethylene signals. Meanwhile, GhRDUF4D expression levels were enhanced after V. dahliae infection. Subsequently, GhRDUF4D was verified by overexpression in Arabidopsis and virus-induced gene silencing treatment in upland cotton. We observed that V. dahliae resistance was significantly enhanced in transgenic Arabidopsis, and weakened in GhRDUF4D silenced plants. This study conducted a comprehensive analysis of the RDUF genes in Gossypium, hereby providing basic information for further functional studies.

Proteo-Trancriptomic Analyses Reveal a Large Expansion of Metalloprotease-Like Proteins in Atypical Venom Vesicles of the Wasp Meteorus pulchricornis (Braconidae)

Meteorus pulchricornis (Ichneumonoidea, Braconidae) is an endoparasitoid wasp of lepidopteran caterpillars. Its parasitic success relies on vesicles (named M. pulchricornis Virus-Like Particles or MpVLPs) that are synthesized in the venom gland and injected into the parasitoid host along with the venom during oviposition. In order to define the content and understand the biogenesis of these atypical vesicles, we performed a transcriptome analysis of the venom gland and a proteomic analysis of the venom and purified MpVLPs. About half of the MpVLPs and soluble venom proteins identified were unknown and no similarity with any known viral sequence was found. However, MpVLPs contained a large number of proteins labelled as metalloproteinases while the most abundant protein family in the soluble venom was that of proteins containing the Domain of Unknown Function DUF-4803. The high number of these proteins identified suggests that a large expansion of these two protein families occurred in M. pulchricornis. Therefore, although the exact mechanism of MpVLPs formation remains to be elucidated, these vesicles appear to be “metalloproteinase bombs” that may have several physiological roles in the host including modifying the functions of its immune cells. The role of DUF4803 proteins, also present in the venom of other braconids, remains to be clarified.

Comparative Genomic Analysis of the DUF34 Protein Family Suggests Role as a Metal Ion Chaperone or Insertase

Members of the DUF34 (domain of unknown function 34) family, also known as the NIF3 protein superfamily, are ubiquitous across superkingdoms. Proteins of this family have been widely annotated as “GTP cyclohydrolase I type 2” through electronic propagation based on one study. Here, the annotation status of this protein family was examined through comprehensive literature review and integrative bioinformatic analyses that revealed varied pleiotropic associations and phenotypes. This analysis combined with functional complementation studies strongly challenges the current annotation and suggests that DUF34 family members may serve as metal ion insertases, chaperones, or metallocofactor maturases. This general molecular function could explain how DUF34 subgroups participate in highly diversified pathways such as cell differentiation, metal ion homeostasis, pathogen virulence, redox and universal stress responses.

A Phosphorus-Limitation Induced, Functionally Conserved DUF506 Protein is a Repressor of Root Hair Elongation in Arabidopsis thaliana

Root hairs (RHs) function in nutrient and water acquisition, root metabolite exudation, soil anchorage and plant-microbe interactions. Longer or more abundant RHs are potential breeding traits for developing crops that are more resource-use efficient and can improve soil health. RH elongation is controlled by both environmental and endogenous factors. While many genes are known to promote RH elongation, relatively little is known about genes and mechanisms that constrain RH growth. Here we demonstrate that a DOMAIN OF UNKNOWN FUNCTION 506 (DUF506) protein, AT3G25240, negatively regulates Arabidopsis thaliana RH growth. The AT3G25240 gene is strongly and specifically induced during P-limitation. Mutants of this gene, which we call REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 (RXR1), have much longer RHs, while over-expression of the gene results in much shorter RHs. RXR1 physically interacts with a Rab-GTPase (RXR2), and an rxr2 mutant phenocopies the rxr1 mutant. Overexpression of a Brachypodium distachyon RXR1 homolog resulted in repression of RH elongation in Brachypodium. Taken together, our results reveal a DUF506-GTPase module with a prominent role in repression of RH elongation that is conserved in monocots and dicots.