The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria uses the type III secretion system (T3SS) to inject effector proteins into cells of its Solanaceous host plants. It is generally assumed that these effectors manipulate host pathways to favor bacterial replication and survival. However, the molecular mechanisms by which type III effectors suppress host defense responses are far from being understood. Based on sequence similarity, Xanthomonas outer protein J (XopJ) is a member of the YopJ/AvrRxv family of SUMO peptidases and acetyltranferases, although its biochemical activity has not yet been demonstrated. Confocal laser scanning microscopy revealed that green fluorescent protein (GFP) fusions of XopJ are targeted to the plasma membrane when expressed in plant cells, which most likely involves N-myristoylation. In contrast to a XopJ(C235A) mutant disrupted in the catalytic triad sequence, the wild-type effector GFP fusion protein was also localized in vesicle-like structures colocalizing together with a Golgi marker protein, suggesting an effect of XopJ on vesicle trafficking. To explore an effect of XopJ on protein secretion, we used a GFP-based secretion assay. When a secreted (sec)GFP marker was coexpressed with XopJ in leaves of Nicotiana benthamiana, GFP fluorescence was retained in reticulate structures. In contrast, in plant cells expressing secGFP alone or along with the XopJ(C235A) mutant, no GFP fluorescence accumulated within the cells. Moreover, coexpressing secGFP together with XopJ led to a reduced accumulation of secGFP within the apoplastic fluid of N. benthamiana leaves, further showing that XopJ affects protein secretion. Transgenic expression of XopJ in Arabidopsis suppressed callose deposition elicited by a T3SS-negative mutant of Pseudomonas syringae pv. tomato DC3000. A role of XopJ in the inhibition of cell wall-based defense responses is discussed.

Intracellular trafficking of viral movement proteins (MPs) in plants has mainly been studied using Tobacco mosaic virus MP30 (TMV MP30) as a model system. Because of the limitations of TMV MP30 expression in Arabidopsis thaliana, these studies have mostly been restricted to tobacco plants. Here we present data on the analysis of transgenic Arabidopsis plants expressing Potato leafroll virus 17-kDa movement protein (MP17) fused to green fluorescent protein. MP17 localizes to secondary branched plasmodesmata (PD) in source but not to simple PD in sink tissues, where MP17 is believed to be degraded by proteolysis. To unravel the intracellular transport path of MP17, we analyzed the relevance of the cytoskeleton and of the secretory pathway on MP17 targeting. To this end, a new incubation system for in vivo analysis of immediate and long-term responses of whole Arabidopsis plants to inhibitor treatments was established. Microscopic and histochemical analysis showed that MP17 is targeted to PD in an actinand endoplasmic reticulum-Golgi-dependent manner. In contrast, degradation of MP17 in sink tissues required intact microtubules and occurred at 26S proteasomes. Interestingly, inhibition of the 26S proteasome led to aggregation of MP17 in aggresome-like structures. Formation of these structures could be inhibited by colchicine, as was shown for aggresomes in mammalian cells.

SUMMARYArabidopsis thaliana contains 18 genes encoding Hsp70s. This heat shock protein superfamily is divided into two sub-families: DnaK and Hsp110/SSE. In order to functionally characterize members of the Hsp70 superfamily, loss-of-function mutants with reduced cytosolic Hsp70 expression were studied. AtHsp70-1 and AtHsp70-2 are constitutively expressed and represent the major cytosolic Hsp70 isoforms under ambient conditions. Analysis of single and double mutants did not reveal any difference compared to wild-type controls. In yeast, SSE protein has been shown to act as a nucleotide exchange factor, essential for Hsp70 function. To test whether members of the Hsp110/SSE sub-family serve essential functions in plants, two members of the sub-family, AtHsp70-14 and AtHsp70-15, were analysed. Both genes are highly homologous and constitutively expressed. Deficiency of AtHsp70-15 but not of AtHsp70-14 led to severe growth retardation. AtHsp70-15-deficient plants were smaller than wild-type and exhibited a slightly different leaf shape. Stomatal closure under ambient conditions and in response to ABA was impaired in the AtHsp70-15 transgenic plants, but ABA-dependent inhibition of germination was not affected. Heat treatment of AtHsp70-15-deficient plants resulted in drastically increased mortality, indicating that AtHsp70-15 plays an essential role during normal growth and in the heat response of Arabidopsis plants. AtHsp70-15-deficient plants are more tolerant to infection by turnip mosaic virus. Comparative transcriptome analysis revealed that AtHsp70-15-deficient plants display a constitutive stress response similar to the cytosolic protein response. Based on these results, AtHsp70-15 is likely to be a key factor in proper folding of cytosolic proteins, and may function as nucleotide exchange factor as proposed for yeast.

Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.

Background and Aims Root hairs are single-cell extensions of the epidermis that face into the soil and increase the root–soil contact surface. Root hairs enlarge the rhizosphere radially and are very important for taking up water and sparingly soluble nutrients, such as the poorly soil-mobile phosphate. In order to quantify the importance of root hairs for maize, a mutant and the corresponding wild type were compared. Methods The rth2 maize mutant with very short root hairs was assayed for growth and phosphorus (P) acquisition in a slightly alkaline soil with low P and limited water supply in the absence of mycorrhization and with ample P supply. Key Results Root and shoot growth was additively impaired under P deficiency and drought. Internal P concentrations declined with reduced water and P supply, whereas micronutrients (iron, zinc) were little affected. The very short root hairs in rth2 did not affect internal P concentrations, but the P content of juvenile plants was halved under combined stress. The rth2 plants had more fine roots and increased specific root length, but P mobilization traits (root organic carbon and phosphatase exudation) differed little. Conclusions The results confirm the importance of root hairs for maize P uptake and content, but not for internal P concentrations. Furthermore, the performance of root hair mutants may be biased by secondary effects, such as altered root growth.