Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.     

Substrate specificity and gene expression of two Penicillium chrysogenum α-l-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54

We previously isolated two α-l-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an “Alpha-L-AF_C” domain in AFQ1 and “ArabFuran-catal” and “AbfB” domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-l-arabinofuranoside and polysaccharides with different specificities. ¹H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and l-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.     

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.     

Heterotrimeric G protein β subunit GPB1 and MAP kinase MPK1 regulate hyphal growth and female fertility in Fusarium sacchari

Heterotrimeric GTP-binding proteins (G proteins) and mitogen-activated protein kinase (MAPK) cascades involve vegetative hyphal growth, development of infection-related structure, colonization in host plant and female fertility in phytopathogenic ascomycete fungi. In this study, a heterotrimeric G protein β subunit (Gβ), GPB1, and MAPK, MPK1, were characterized from Fusarium sacchari (= Gibberella sacchari; mating population B of the G. fujikuroi-species complex). GPB1 and MPK1 showed high homology to known Gβ and Fus3/Kss1 MAP kinases of other filamentous ascomycetes, respectively. Disruption (Δ) of gpb1 suppressed hyphal branching and accelerated aerial hyphae formation in F. sacchari. Oppositely, disruption of mpk1 caused delayed aerial hyphae formation. These indicated that GPB1 regulates vegetative hyphal growth negatively, and MPK1 does positively in F. sacchari. Both Δgpb1 and Δmpk1 showed female sterility. Level of intracellular cAMP in Δgpb1 was lower than wild type. Exogenous cyclic AMP (cAMP) partially restored enhanced aerial hyphae formation. These suggested that abnormal hyphal growth was caused by depletion of intracellular cAMP in Δgpb1. cAMP has been reported to suppress development of perithecia in crossing between wild type strains. Thus, precise regulation of intracellular cAMP level via Gβ/MAPK is essential for normal hyphal growth and fertility.     

Cellular level observation of water loss and the refilling of tracheids in the xylem of Cryptomeria japonica during heartwood formation

The mechanism of heartwood formation in Cryptomeria japonica D. Don has long been studied since heartwood formation is a fundamental physiological feature of trees. In this study, the water distribution in the xylem of C. japonica was investigated at the cellular level to reveal the role of water distribution in the xylem during heartwood formation. Samples were taken from different heights of each trunk, in which the phases of heartwood formation differed. These were designated as SIH, which consisted of sapwood, intermediate wood, and heartwood; SI, which consisted of sapwood and intermediate wood but no heartwood; and S-all, which consisted entirely of sapwood. Cryo-scanning electron microscopic observations of the heartwood-formed (SIH) and non-heartwood-formed (SI and S-all) xylem revealed different patterns of water distribution changes in tracheids between the latewood and earlywood. In the latewood, almost all tracheids were filled with water in all areas from the sapwood to the heartwood (98–100% of tracheids had water in their lumina). In the earlywood, however, the water distribution differed between the sapwood (95–99%), intermediate wood (7–12%), and heartwood (4–100%). Many of the tracheids in the xylem, where the sapwood changed to intermediate wood lost water. In the heartwood, some tracheids remained empty, while others were refilled with water. These results suggest that the water distribution changes in individual tracheids are closely related to heartwood formation. Water loss from tracheids may be an important factor inducing heartwood formation in the xylem of C. japonica.     

Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation

The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.