Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice.     

Frequency and coverage of trinucleotide repeats in eukaryotes

In the aim to assess whether the tri-repeat shortage reported in vertebrates affects specific motifs, such as those causing neuromuscular diseases in man, we detected approximate di-, tri- and tetra-repeats (STR) longer than 25 bases in human chromosomes 21 and 22, and in some model organisms (M. musculus, D. melanogaster, C. elegans, A. thaliana and S. cerevisiae). We found that overall STR are more represented in mouse and in man than in the other organisms. However, tri-repeats are less represented than di- and tetra- in man and mouse, but show intermediate values between di- and tetra- in the other organisms. In man, ACG shows the lowest both frequency and coverage, ATC the highest coverage and AAT the highest frequency. In general, coverage and frequency of tri-repeats are linearly related, except for ACC, ATC, AAG, AGG motifs in man and AAG, AGG in mouse, which exhibit unexpectedly long repeats. Often their copy numbers exceed that found responsible for the dynamic mutations, set at around 40. The shortage in frequency and coverage of tri- vs. di- and tetra-repeats observed in man and mouse can be ascribed to a subset of the remaining tri-repeat motifs, but among them those recognized as dynamically mutable (AAG, AGC and CCG) are not the least represented. Possible constraints in tri-repeat expansion seem to be structural and conserved along the evolutionary scale: a motif-specific relaxation of the relevant controls may be responsible for the occasional expansions found in mouse and man.     

The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.     

The complexity of targeting EGFR signalling in cancer: from expression to turnover

The epidermal growth factor receptor (ErbB1 or EGFR) has been found to be altered in a variety of human cancers. A number of agents targeting these receptors, including specific antibodies directed against the ligand-binding domain of the receptor and small molecules that inhibit kinase activity are either in clinical trials or are already approved for clinical treatment. However, identifying patients that are likely to respond to such treatments has been challenging. As a consequence, it still remains important to identify additional alterations of the tumor cell that contribute to the response to EGFR-targeted agents. While EGFR-mediated signalling pathways have been well established, there is still a rather limited understanding of how intracellular protein-protein interactions, ubiquitination, endocytosis and subsequent degradation of EGFR contribute to the determination of sensitivity to EGFR targeting agents and are emerging areas of investigation. This review primarily focuses on the basic signal transduction pathways mediated through activated membrane bound and/or endosomal EGFR and emphasizes the need to co-target additional proteins that function either upstream or downstream of EGFR to improve cancer therapy.     

Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells.

This study demonstrates how the potentiating effects of E2 on insulin signaling in ER-positive breast cancer cells are consequent to an enhanced IRS-1 expression [corrected]. It induces an increase of both PI-3K/AKT and ERK1/2 activities. A direct action of E2 in the regulating mouse IRS-1 gene is also investigated in both Chinese hamster ovary and MCF-7 cells that are transfected with mouse IRS-1 regulatory sequences. The authors have reported, for the first time, how E2 induction of IRS-1 mRNA was correlated with a direct positive regulatory role of E2 on the IRS-1 promoter. This effect seems to be not strictly related to the cell type.     

Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches： （i） AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain （GVG） chemical-inducible expression system; （ii） deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and （iii） suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element （HPT-Ds）, We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community,     

RBS1, an RNA Binding Protein,Interacts with SPIN1 and Is Involved in Flowering Time Control in Rice

The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.     

BKV QPCR detection and infection monitoring in renal transplant recipients

BKV associated nephropathy (BKVAN) is a cause of renal dysfunction and loss of the graft in transplants. Viral primary infection is usually inapparent and then BKV establishes latency in kidneys. Reactivation occurs in immunocompromised conditions in renal transplant recipients who can develop a subclinical nephritis and eventually a BKV-associated interstitial nephritis or a BKVAN. In this study, we searched for BKV copies in urine and plasma of renal transplants by quantitative assay (QPCR). Results showed that in several patients clearance of viremia is associated with persistent viruria, suggesting that both specimens are necessary to correctly monitor a BKVAN.