Genetic interaction between AINTEGUMENTA (ANT) and the ovule identity genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2)

AINTEGUMENTA (ANT) promotes initiation and growth of ovule integuments which cell fate is specified by ovule identity factors, such as SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2). To study the genetic interaction between ANT and the ovule identity genes, we have obtained a stk shp1 shp2 ant quadruple mutant. The molecular and morphological characterization of the quadruple mutant and its comparison with the stk shp1 shp2 triple mutant, the shp1 shp2 ant triple mutant and the stk ant double mutant are here presented.     

Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis

In Arabidopsis thaliana and many other plant species, ovules arise from carpel tissue as new meristematic formations. Cell fate in proliferating ovule primordia is specified by particular ovule identity factors, such as the homeodomain factor BELL1 (BEL1) and MADS box family members SEEDSTICK (STK), SHATTERPROOF1 (SHP1), SHP2, and AGAMOUS. Both in the bel1 mutant and the stk shp1 shp2 triple mutant, integuments are transformed into carpelloid structures. Combining these mutants in a bel1 stk shp1 shp2 quadruple mutant, we showed that the bel1 phenotype is significantly enhanced. We also demonstrate that ovule differentiation requires the regulation of the stem cell maintenance gene WUSCHEL, repression of which is predominantly maintained by BEL1 during ovule development. Based on yeast three-hybrid assays and genetic data, we show that BEL1 interacts with the ovule identity MADS box factors when they dimerize with SEPALLATA proteins. We propose a model for ovule development that explains how the balance between carpel identity activity and ovule identity activity is established by a MADS box homeodomain protein complex.     

A new role for the SHATTERPROOF genes during Arabidopsis gynoecium development

Gynoecium development is a complex process which is regulated by key factors that control the spatial formation of the apical, medial and basal parts. SHATTERPROOF1 (SHP1) and SHP2, two closely related MADS-box genes, redundantly control the differentiation of the dehiscence zone and promote the lignification of adjacent cells. Furthermore, SHP1 and SHP2 have shown to play an important role in ovule identity determination. The present work identifies a new function for these two genes in promoting stigma, style and medial tissue development. This new role was discovered by combining the shp1 shp2 double mutant with the aintegumenta (ant) and crabs claw (crc) mutants. In quadruple mutant flowers, the inner whorl is composed of unfused carpels which lack almost completely apical and medial tissues, a phenotype similar to the previously reported fil ant and lug ant double mutants.     

The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice

Genes that control ovule identity were first identified in Petunia. Co-suppression of both FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, two D-lineage genes, resulted in the homeotic transformation of ovules into carpelloid structures. Later in Arabidopsis it was shown that three genes, SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK), redundantly control ovule identity, because in the stk shp1 shp2 triple mutant ovules lose identity and are transformed into carpel and leaf-like structures. Of these three Arabidopsis genes STK is the only D-lineage gene, and its expression, like FBP7 and FBP11, is restricted to ovules. OsMADS13 is the rice ortholog of STK, FBP7, and FBP11. Its amino acid sequence is similar to the Arabidopsis and Petunia proteins, and its expression is also restricted to ovules. We show that the osmads13 mutant is female sterile and that ovules are converted into carpelloid structures. Furthermore, making carpels inside carpels, the osmads13 flower is indeterminate, showing that OsMADS13 also has a function in floral meristem determinacy. OsMADS21 is most likely to be a paralog of OsMADS13, although its expression is not restricted to ovules. Interestingly, the osmads21 mutant did not show any obvious phenotype. Furthermore, combining the osmads13 and the osmads21 mutants did not result in any additive ovule defect, indicating that osmads21 does not control ovule identity. These results suggest that during evolution the D-lineage gene OsMADS21 has lost its ability to determine ovule identity.     

MADS-box protein complexes control carpel and ovule development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.     

The MADS box genes SEEDSTICK and ARABIDOPSIS Bsister play a maternal role in fertilization and seed development

The haploid generation of flowering plants develops within the sporophytic tissues of the ovule. After fertilization, the maternal seed coat develops in a coordinated manner with formation of the embryo and endosperm. In the arabidopsis bsister (abs) mutant, the endothelium, which is the most inner cell layer of the integuments that surround the haploid embryo sac, does not accumulate proanthocyanidins and the cells have an abnormal morphology. However, fertility is not affected in abs single mutants. SEEDSTICK regulates ovule identity redundantly with SHATTERPROOF 1 (SHP1) and SHP2 while a role in the control of fertility was not reported previously. Here we describe the characterization of the abs stk double mutant. This double mutant develops very few seeds due to both a reduced number of fertilized ovules and seed abortions later during development. Morphological analysis revealed a total absence of endothelium in this double mutant. Additionally, massive starch accumulation was observed in the embryo sac. The phenotype of the abs stk double mutant highlights the importance of the maternal-derived tissues, particularly the endothelium, for the development of the next generation.     

Fleshy Fruit Expansion and Ripening Are Regulated by the Tomato SHATTERPROOF Gene TAGL1

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.     

Shp1, a regulator of protein phosphatase 1 Glc7, has important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans

In yeast, the type 1 protein phosphatase (PP1) catalytic subunit Glc7 is involved in the regulation of multiple cellular processes and thought to achieve specificity through association with different regulatory subunits. Here, we report that the Glc7 regulator Shp1 plays important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans. SHP1 deletion caused the formation of rod-shaped yeast cells with slow growth. Flow cytometry analysis revealed that shp1Δ cells showed a prolonged G(2)/M phase, which was rescued by deleting the spindle-checkpoint gene MAD2. Furthermore, shp1Δ cells were hypersensitive to heat and genotoxic stresses. Interestingly, depletion of Glc7 caused defects similar to the shp1Δ mutant such as arrest at G(2)/M transition; and the GLC7/glc7Δ heterozygous mutant exhibited increased sensitivity to genotoxic stresses, consistent with the recent finding that Saccharomyces cerevisiae Glc7 has a role in DNA damage response. We also show that Shp1 is required for the nuclear accumulation of Glc7, suggesting that Shp1 executes its cellular function partly by regulating Glc7 localization.     

The Tyrosine 343 Residue of Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Is Important for Its Interaction with SHP1, a Cytoplasmic Tyrosine Phosphatase with Tumor Suppressor Functions

The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK^K210R mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr³³⁸, Tyr³⁴², and Tyr³⁴³) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr³³⁸ or Tyr³⁴² did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr³⁴³ abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK^Y343F mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK^Y343F. In conclusion, we identified Tyr³⁴³ of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

Penetration and Post-Penetration Resistance to Magnaporthe oryzae in Arabidopsis

The rate of entry of Magnaporthe oryzae into the Arabidopsis pen2 quintuple (pen2 NahG pmr5 agb1 mlo2) mutant was significantly higher than those into the pen2 quadruple (pen2 NahG pmr5 agb1 and pen2 NahG pmr5 mlo2) mutants. The lengths of the infection hyphae in the pen2 quintuple mutant were intermediate between the pen2 quadruple mutants. These results suggest that different genetic networks, consisting of PEN2, PMR5, AGB1, and MLO2, control penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

The Budding Yeast Cdc48Shp1 Complex Promotes Cell Cycle Progression by Positive Regulation of Protein Phosphatase 1 (Glc7)

The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48^Shp1 complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B) kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48^Shp1 complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.     

胶孢炭疽菌C2H2型转录因子CgGcp1的生物学功能

【背景】胶孢炭疽菌(Colletotrichum gloeosporioides)可以寄生于多种植物,侵染方式多样,能够引起严重的农业危害。在胶孢炭疽菌中,CgGcp1是一个C2H2型的转录因子,关于其生物学功能的研究未见报道。【目的】明确CgGcp1的生物学功能,为深入解析该病菌的致病机制奠定一定的理论依据。【方法】构建CgGCP1基因的敲除载体,利用同源重组得到敲除突变体。通过表型分析,包括营养生长、胁迫响应、孢子产生、附着胞形成及致病性分析等,明确该基因的生物学功能。【结果】CgGCP1基因敲除突变体生长速率较野生型减慢,对SDS、刚果红、NaCl和甘油更加敏感,孢子产量显著降低,附着胞的形成率降低且侵入能力减弱,在橡胶叶片上的致病力明显下降。【结论】CgGcp1参与调控胶孢炭疽菌营养生长、细胞壁完整性、分生孢子产生、附着胞形成与侵入和致病性。     

SH2 domain-containing phosphatase 2 is a critical regulator of connective tissue mast cell survival and homeostasis in mice

Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT → SHP2 → Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.